Monkey alpha-7 nicotinic acetylcholine receptor and methods of use thereof

ABSTRACT

Monkey alpha-7 neuronal nicotinic acetylcholine receptor polypeptides, as well as the DNA (RNA) encoding such polypeptides, are disclosed. Also disclosed are methods for utilizing such polypeptides in diagnostic assays for identifying mutations in nucleic acid sequences encoding the polypeptides of the present invention, for detecting altered levels of the polypeptide of the present invention as a means of detecting diseases and methods of screening potential modulators of the novel alpha-7 receptor disclosed herein. Transgenic animals expressing polypeptides disclosed herein are also described.

The present application claims the benefit of U.S. ProvisionalApplication Ser. Nos. 60/447,288, filed Feb. 14, 2003, and 60/453,204,filed Mar. 11, 2003, which are hereby incorporated by reference in theirentirety.

BACKGROUND OF THE INVENTION

There are two types of receptors for the neurotransmitter,acetylcholine: muscarinic receptors and nicotinic receptors, based onthe selectivity of action of muscarine and nicotine, respectively.Muscarinic receptors are G-protein coupled receptors. Nicotinicreceptors are members of the ligand-gated ion channel family. Whenactivated, the conductance of ions across the nicotinic ion channelsincreases.

Nicotinic alpha-7 receptor protein forms a homo-pentameric channel invitro that is highly permeable to a variety of cations (e.g., Ca⁺⁺).Each nicotinic alpha-7 receptor has four transmembrane domains, namedM1, M2, M3, and M4. The M2 domain has been suggested to form the walllining the channel. Sequence alignment shows that nicotinic alpha-7 ishighly conserved during evolution. The M2 domain that lines the channelis identical in protein sequence from chicken to human. For discussionsof the alpha-7 receptor, see, e.g., Revah et al. (1991), Nature, 353,846-849; Galzi et al. (1992), Nature 359, 500-505; Fucile et al. (2000),PNAS 97(7), 3643-3648; Briggs et al. (1999), Eur. J. Pharmacol. 366(2-3), 301-308; and Gopalakrishnan et al. (1995), Eur. J. Pharmacol.290(3), 237-246.

The nicotinic alpha-7 receptor channel is expressed in various brainregions and is believed to be involved in many important biologicalprocesses in the central nervous system (CNS), including learning andmemory. Nicotinic alpha-7 receptors are localized on both presynapticand postsynaptic terminals and have been suggested to be involved inmodulating synaptic transmission. Nicotinic alpha-7 receptors aretherefore of interest in drug development of compounds that modulate theactivity of neuronal nicotinic alpha-7 receptors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Nucleotide sequence for a cDNA encoding for rhesus monkeyalpha-7 nACh receptor subunit. (SEQ ID NO: 1).The start codon ATG andstop codon TAA are indicated in bold.

FIG. 2: Amino acid sequence for rhesus monkey alpha-7 nACh receptorsubunit. (SEQ ID NO: 2)

FIG. 3: Nucleotide sequence for a cDNA encoding for mutant (L270T)rhesus monkey alpha-7 nACh receptor subunit. (SEQ ID NO: 3) The startcodon ATG and stop codon TAA are indicated in bold.

FIG. 4: Amino acid sequence for mutant (L270T) rhesus monkey alpha-7nACh receptor subunit. (SEQ ID NO: 4)

FIG. 5: Nucleotide sequence for a cDNA encoding for double-mutant(L270T/S193N) for rhesus monkey alpha-7 nACh receptor subunit. The startcodon ATG and stop codon TAA are indicated in bold. (SEQ ID NO: 5)

FIG. 6: Amino acid sequence for double-mutant (L270T/S193N) for rhesusmonkey alpha-7 nACh receptor subunit. (SEQ ID NO: 6)

FIG. 7: Functional dose-dependent response of nicotine and GTS-21 tomutant (L270T) alpha7 nAchR stably expressed in QM7 cells. Theexperiment was measured with FLIPR. The EC50 of GTS-21, a specificalpha7 nAchR agonist, is better or comparable to that of nicotine.

FIG. 8: Dose response curve of rhesus monkey wild-type alpha-7 clone #1to acetylcholine (Ach). The EC₅₀ of this receptor is 38 μM, which iscomparable to EC₅₀s for human and rat wild type alpha-7 receptors (21 μMand 28 μM respectively; Papke, R. L. and Porter, J. K. (2002)Comparative pharmacology of rat and human alpha7 nAChR conducted withnet charge analysis. Br. J. Pharmacol. 137(1), 49-61.)

FIG. 9: Dose-response curve for double-mutant (L270T/S193N) for rhesusmonkey alpha-7 nACh receptor subunit using GTS-21.

DESCRIPTION OF THE INVENTION

The present invention relates to monkey alpha-7 neuronal nicotinicacetylcholine receptor (“alpha-7 receptor”), variants, fragmentsthereof, antibodies thereto, their uses, etc. One aspect of theinvention is an isolated full-length rhesus monkey alpha-7 receptorprotein, as represented by FIG. 2 (SEQ ID NO: 2) (wild-type), andmutations to it, such as amino acid substitutions in the M2 domain.Examples of variants, include, e.g., the polypeptide sequences shown inFIGS. 4 and 6 (SEQ ID NOS:4 and 6). The polypeptides represented bythese sequences each have 502 amino acids.

Another aspect of the invention is an isolated cDNA, which encodes afull-length rhesus monkey alpha-7 receptor protein. Typical cDNAs arerepresented by SEQ ID NO: 1 (FIG. 1; wild-type) and SEQ ID NOS: 3 and 5(FIGS. 3 and 5; mutant). The plasmid MKALPHA7 containing the cDNA of SEQID NO:1 was deposited on Feb. 13, 2003, with the American Type CultureCollection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209,U.S.A. under the provisions of the Budapest Treaty for the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure and was accorded ATCC Accession No. PTA-5004.

Thus, the invention relates, e.g., to an isolated polynucleotidecomprising the cDNA sequence of FIG. 1, 3, or 5 (SEQ ID NOS 1,3, or 5).The invention also relates to an isolated polynucleotide comprising afragment or variant of these sequences, or complements thereto.

Another aspect of the invention is an isolated polynucleotide whichcomprises a nucleotide sequence that codes without interruption for thepolypeptide of FIG. 2, 4, or 6 (SEQ ID NO: 2, 4, or 6) or a fragment,variant, or complement thereof. A polynucleotide which “codes withoutinterruption” refers to a polynucleotide having a continuous openreading frame (“ORF”) as compared to an ORF which is interrupted byintrons or other noncoding sequences.

The invention also relates to methods of making the above-describedpolypeptides and fragments thereof, or polynucleotides. The methodsinclude,e.g., methods of making constructs which comprise and/or expressthe polynucleotide sequences; and methods of transforming cells withconstructs capable of expressing the polypeptides, culturing thetransformed cells under conditions effective to express thepolypeptides, and harvesting (recovering) the polypeptides; toantibodies, antigen-specific fragments, or other specific bindingpartners which are specific (selective) for the polypeptides; to methodsof detecting a disease condition or a susceptibility to a diseasecondition that is associated with aberrant expression (e.g., under- orover-expression) of the polypeptides or polynucleotides, or with variantforms (e.g., mutants, polymorphisms, SNPs, etc.) of the polypeptides orpolynucleotides; to methods of treating such disease conditions (e.g.,any of a variety of memory dysfunctions) or of stimulating memoryformation; to methods of using polypeptides, polynucleotides orantibodies of the invention to detect the presence or absence, and/or toquantitate the amounts, of the polypeptides and polynucleotides of theinvention in a sample; to methods of detecting mutations in thepolypeptide or polynucleotide sequences which are associated with adisease condition; to methods of using the polypeptides orpolynucleotides, or cells transformed with the polynucleotides, toscreen for potential therapeutic agents, e.g., agents which modulate theactivity or amounts of the polynucleotides or polypeptides; totransgenic animals which express the polypeptides or knockout animalswhich do not express the polypeptides; or for other potential uses.

For example, the invention relates to an isolated polypeptide,comprising the amino acid sequence of FIG. 2, 4, or 6 (SEQ ID NOS: 2, 4,or 6), or a fragment or variant thereof. The polypeptide may comprise,e.g., at least about 10, 12, 14, 15, 20, 25, 50, 100, 200, etc.,contiguous amino acids of FIG. 2, 4, or 6 (SEQ ID NOS: 2, 4, or 6)and/or may have a sequence identity of, e.g., at least about 65%,70-75%, 80-85%, 90-95% or 97-99% to FIG. 2, 4, or 6 (SEQ ID NOS: 2, 4,or 6) or a fragment thereof; and/or may comprise a sequence that issubstantially homologous to FIG. 2, 4, or 6 (SEQ ID NOS: 2, 4, or 6) ora fragment thereof; and/or may be encoded by cDNA contained in ATCCDeposit No PTA-5004, or a fragment thereof. The polypeptide may furthercomprise a heterologous sequence; may exhibit alpha-7 receptor activity;may be from a mammal, and/or may be substantially purified. Thepolypeptide may have the amino acid sequence of FIG. 2, 4, or 6 (SEQ IDNOS: 2, 4, or 6).

In another aspect, the invention relates to an isolated polynucleotidewhich comprises the nucleotide sequence of FIG. 1, 3, or 5 (SEQ ID NOS:1, 3, or 5) or a fragment or variant of FIG. 1, 3, or 5 (SEQ ID NOS: 1,3, or 5) or a complement thereof The polynucleotide many comprise; e.g.,at least about 8, 10, 12, 14, 15, 20, 25, 30, 50, etc., contiguousnucleotides of FIG. 1, 3, or 5 (SEQ ID NOS: 1, 3, or 5), e.g., about 15continuous nucleotides. The polynucleotide may further comprise aheterologous sequence; and/or may be from a mammal, and/or may be DNA,cDNA, RNA, PNA or combinations thereof. The polynucleotide may have anucleotide sequence of the cDNA contained in ATCC Deposit No. PTA-5004or of a fragment thereof; and/or may comprise a sequence that hybridizesto FIG. 1, 3, or 5 (SEQ ID NOS: 1, 3, or 5) or a fragment thereof underconditions of high stringency; and/or may comprise a sequence that issubstantially homologous to FIG. 1, 3, or 5 (SEQ ID NOS: 1, 3, or 5) ora fragment thereof; and/or may have a sequence identity of, e.g., atleast about 65%, 70-75%, 80-85%, 90-95% or 97-99% to FIG. 1, 3, or 5(SEQ ID NOS: 1, 3, or 5) or a fragment thereof; and/or may have thenucleotide sequence of FIG. 1, 3, or 5 (SEQ ID NOS: 1, 3, or 5).

In another aspect, the invention relates to a recombinant constructcomprising a polynucleotide as above, which may be operatively linked toa regulatory sequence, e.g., wherein said construct comprises abaculovirus expression vector. The invention also relates to a cellcomprising such a construct, e.g., a mammalian, human, yeast, QM7, QT6,or insect cell, preferably an SF9 cell. The invention also relates to amethod of making such a cell, comprising introducing a construct orpolynucleotide as above into a cell. The invention also relates to amethod to make a polypeptide of the invention, comprising incubating acell as above under conditions in which the polypeptide is expressed,and harvesting the polypeptide.

In another aspect, the invention relates to an antibody,antigen-specific antibody fragment, or other specific binding partner,which is specific for a polypeptide of the invention, e.g., wherein saidantibody, antigen-specific antibody fragment, or specific bindingpartner is specific for the polypeptide of SEQ ID NO: 2 or 4 or afragment or variant thereof.

In another aspect, the invention relates to methods of diagnosis, e.g.,a method to determine the presence of a disease condition or asusceptibility to a disease condition in a patient in need thereof,where said condition is associated with an over- or underexpression of apolynucleotide (e.g., mRNA) of the invention, comprising contacting acell, tissue, cell extract, or nucleic acid of said patient with apolynucleotide as above, and/or determining the amount or level of saidnucleic acid. The cell or nucleic acid may be from the brain of saidpatient, e.g., from the hippocampus, and may be from a neuron.

The invention also relates to a method of diagnosis, comprisingdetermining a mutation or polymorphism or SNP in the genome of a cell,wherein said mutation occurs in the nucleotide sequence of FIG. 1, 3, or5 (SEQ ID NOS: 1, 3, or 5), or in the sequence of a polynucleotide whichencodes a polypeptide of FIG. 2, 4, or 6 (SEQ ID NOS: 2, 4, or 6).

The invention also relates to a method to determine the presence of adisease condition or a susceptibility to a disease condition, whereinsaid condition is associated with an over- or under-expression of, oractivity of, a polypeptide of the invention, comprising contacting acell, tissue or cell extract of said patient with an antibody which isspecific for a polypeptide of the invention, and detecting the amount oractivity of said polypeptide.

The invention also relates to a method to determine the presence of adisease condition or susceptibility to a disease condition, wherein saidcondition is associated with a mutated nicotinic alpha-7 receptor,comprising identifying such a mutation in a nicotinic alpha-7 receptorisolated from a patient.

In another aspect, the invention relates to methods to screen for agentsthat modulate (e.g., stimulate or inhibit) expression or activity of apolypeptide of the invention, or of a polynucleotide which encodes it,comprising contacting a cell, preferably from neuronal tissue, or atissue cell extract with a putative modulatory agent, and measuring theamount or activity of said polypeptide or polynucleotide, including,e.g., monitoring net charge flow through the alpha-7 receptor/channel inresponse to said agent, measuring the total amount of charge flowingacross the membrane of said cell, or measuring the change in acalcium-sensitive dye present in said cell, in response to said agent.

The invention also relates to methods to screen for agents which bind toa polypeptide or polynucleotide of the invention, comprising contactingan inventive polypeptide or polynucleotide with a putative binding agentand determining the presence of a bound complex (e.g., a nucleic acidhybrid, antigen-antibody complex, protein-protein interaction,ligand-target complex, or the like). Methods of the invention can beperformed in vitro, ex vivo, or in vivo.

In another aspect, the invention relates to a transgenic animal (e.g., amouse), preferably a non-human mammal, comprising at least one copy ofan alpha-7 receptor polynucleotide of the invention, wherein the animaloverexpresses functional alpha-7 receptor, or a functional fragment oranalog thereof, compared to a non-transgenic animal. In another aspect,the invention relates to a knockout animal, e.g., a primate, whosegenome lacks a gene expressing a functional alpha-7 receptor orfunctional fragment or variant thereof; or to a transgenic animal inwhich the natural alpha-7 receptor is replaced by a heterologoustransgenic. Such transgenic animals can have a modified response to anicotinic receptor ligand, e.g., altered desensitization properties.

In another aspect, the invention relates to a pharmaceutical compositioncomprising a polypeptide or polynucleotide of the invention and apharmaceutically acceptable carrier. In another aspect, the inventionrelates to a prophylactic or therapeutic method of treating a diseasecondition mediated by, or associated with, aberrant expression and/oractivity of the alpha-7 receptor, comprising administering to a patientin need thereof an agent which modulates the expression and/or activityof said alpha-7 receptor.

Polypeptides

A polypeptide of the present invention may be a recombinant polypeptide,a natural polypeptide or a synthetic or semi-synthetic polypeptide, orcombinations thereof, preferably a recombinant polypeptide. As usedherein, the terms polypeptide, oligopeptide and protein areinterchangeable. A nicotinic alpha-7 receptor channel polypeptide of thepresent invention can comprise various domains, including, e.g., asignal sequence at about amino acid positions 1-22, and at least fourtransmembrane domains: M1 at about amino acid positions 231-255, M2 atabout amino acid positions 262-283, M3 at about amino acid positions293-315, and M4 at about amino acid positions 470-494. This numbering isin accordance with the amino acid sequence set forth in FIG. 2 (SEQ IDNO:2).

The polypeptides of the present invention are preferably provided in anisolated form, and may be purified, e.g. to homogeneity. The term“isolated,” when referring, e.g., to a polypeptide or polynucleotide,means that the material is removed from its original environment (e.g.,the natural environment if it is naturally occurring), and isolated orseparated from at least one other component with which it is naturallyassociated. For example, a naturally-occurring polypeptide present inits natural living host is not isolated, but the same polypeptide,separated from some or all of the coexisting materials in the naturalsystem, is isolated. Such polypeptides could be part of a composition,and still be isolated in that such composition is not part of itsnatural environment.

The terms “fragment” or “variant,” when referring to a polypeptide ofthe invention, mean a polypeptide which retains substantially at leastone of the biological functions or activities of the polypeptide. Such abiological function or activity can be, e.g., any of those describedabove, and includes having the ability to react with an antibody, i.e.,having a epitope-bearing peptide. Fragments or variants of thepolypeptide of FIGS. 2, 4, or 6 (SEQ ID NOS: 2, 4, or 6) have sufficientsimilarity to FIG. 2, 4, or 6 (SEQ ID NOS: 2, 4, or 6) so that at leastone activity (e.g., an activity expressed by a functional domainthereof, or the ability to react with an antibody or antigen-bindingfragment of the invention) of the native polypeptides is retained.Biological functions or activities of alpha-7 nicotinic acetylcholinereceptor polypeptide, which forms a homo-pentameric channel in vitro orwhen expressed in cells, include nicotinic ligand-binding andligand-gated ion conductance, e.g., the channel is selectively permeableto Ca²⁺ and other cations, when activated by a receptor agonist.Polypeptide fragments of the invention may be of any size that iscompatible with the objects of the invention. They may range in sizefrom the smallest specific epitope (e.g., about 6 amino acids) to anearly full-length gene product (e.g., a single amino acid shorter thanthe polypeptides sequences in FIG. 2, 4, or 6 (SEQ ID NOS: 2, 4, or 6).A preferred fragment that would still retain biological functionexcludes the signal peptide of the sequence (for example, signalsequence may include amino acids 1-22 of FIG. 2, 4, or 6 (SEQ ID NOS: 2,4, or 6)).

Fragments of the polypeptides of the present invention may be employed,e.g., for producing the corresponding fill-length polypeptide by peptidesynthesis, e.g., as intermediates for producing the fill-lengthpolypeptides; for inducing the production of antibodies orantigen-binding fragments; as “query sequences” for the probing ofpublic databases, or the like.

A variant of a polypeptide of the invention may be, e.g., (i) one inwhich one or more of the amino acid residues are substituted with aconserved or non-conserved amino acid residue (preferably a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code, or (ii) one in which one or moreof the amino acid residues includes a substituent group, or (iii) one inwhich the polypeptide is fused with another compound, such as a compoundto increase the half-life of the polypeptide (for example, polyethyleneglycol), or (iv) one in which additional amino acids are fused to thepolypeptide, such as a leader or secretory sequence or a sequence whichis employed for purification of the polypeptide, commonly for thepurpose of creating a genetically engineered form of the protein that issusceptible to secretion from a cell, such as a transformed cell. Theadditional amino acids may be from a heterologous source, or may beendogenous to the natural gene.

Variant polypeptides belonging to type (i) above include, e.g., mutants,analogs and derivatives. A variant polypeptide can differ in amino acidsequence by, e.g., one or more additions, substitutions, deletions,insertions, inversions, fusions, and truncations or a combination of anyof these.

In one embodiment, the monkey nicotinic alpha-7 acetylcholine receptorsubunit comprises at least one mutation. Preferably, the mutation is inthe M2 domain of the subunit. The mutation can result in slowerdesensitization than in the wild type nicotinic alpha-7 polypeptide,enhancing Ca⁺⁺ assay read-outs. Desensitization can be determinedroutinely, e.g., using the methods described in the examples, or in thepublications cited herein. M2 domains (generally about 20 amino acids inlength) of the alpha-7 polypeptides have been characterized from avariety of species and have been shown to play an important role in ionpermeation through nicotinic alpha-7 receptor channels. See, e.g.,Changeux et al. (1992) Q. Rev. Biophys. 25, 395-432; Bertrand et al.(1993) Proc. Natl. Acad. Sci. 90, 6971-6975; and Revah et al. (1991)Nature 353, 846-849. Examples of mutations in the M2 domain include, forexample, Leu at position 270 substituted with Thr (L270T) and Val atposition 274 substituted with Thr (V274T). Other mutants include thosesingle and double monkey nicotinic alpha-7 mutations equivalent to thesingle and double chicken nicotinic alpha-7 mutations L247T, L247S,L247F, L247V, V251T, T244Q, E237A/V251T, E237A/L247T, E237A/L247S,E237A/L247V, E237A/L247F, E237A/T244Q, E237A/L254T, or E237A/L255T. Theequivalent single and double monkey nicotinic alpha-7 mutations are,respectively, L270T, L270S, L270F, L270V, V274T, T267Q, E260A/V274T,E260A/L270T, E260A/L270S, E260A/L270V, E260A/L270F, E260A/T267Q,E260A/L277T, or E260A/L278T. In addition, other residues, when mutated,may also lead to slower desensitization, e.g., residues that face thechannel lumen and are aligned along the meridian of an alpha-helix. See,e.g., Bertrand et al. (1995) The Neurosciences 7, 75-90 and Bertrand etal. (1993), Current Opinion in Cell Biology 5, 688-693.

In addition to mutations in the M2 region, mutations can be made inother regions or domains of the polypeptide, and the correspondingpolynucleotide sequences which encode it. This includes one or moremutations, such as: A65V, RI56W, S193N, K208R, K208S, M395V, A398Vand/or A398T. These mutations are preferably made in combination withmutations in the M2 region, and therefore include double-mutants,triple-mutants, etc. FIGS. 5 and 6 (SEQ ID NOS: 5 and 6) show an exampleof a double-mutant comprising amino acid substitutions in the M2 region(L270T) and in an other region (S193N).

Variant polypeptides belonging to type (ii) above include, e.g.,modified polypeptides. Known polypeptide modifications include, but arenot limited to, glycosylation, acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphatidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent crosslinks, formation ofcystine, formation of pyroglutamate, formylation, gamma carboxylation,glycosylation, GPI anchor formatin, hydroxylation, iodination,methylation, myristoylation, oxidation, proteolytic processing,phosphorylation, prenylation, racemization, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to proteins such asarginylation, and ubiquitination.

Such modifications are well-known to those of skill in the art and havebeen described in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in many basic texts,such as Proteins—Structure and Molecular Properties, 2nd ed., T. E.Creighton, W. H. Freeman and Company, New York (1993). Many detailedreviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (1990) Meth.Enzymol. 182:626-646 and Rattan et al. (1992) Ann. N.Y. Acad. Sci.663:48-62.

Variant polypeptides belonging to type (iii) are well-known in the artand include, e.g., PEGulation or other chemical modifications.

Variants polypeptides belonging to type (iv) above include, e.g.,preproteins or proproteins which can be activated by cleavage of theproprotein portion to produce an active mature polypeptide. Variantsinclude a variety of hybrid, chimeric or fusion polypeptides. Typicalexample of such variants are discussed elsewhere herein.

Many other types of variants are known to those of skill in the art. Forexample, as is well known, polypeptides are not always entirely linear.For instance, polypeptides may be branched as a result ofubiquitination, and they may be circular, with or without branching,generally as a result of post-translation events, including naturalprocessing events and events brought about by human manipulation whichdo not occur naturally. Circular, branched and branched circularpolypeptides may be synthesized by non-translational natural processesand by synthetic methods.

Modifications or variations can occur anywhere in a polypeptide,including the peptide backbone, the amino acid side-chains and the aminoor carboxyl termini. The same type of modification may be present in thesame or varying degree at several sites in a given polypeptide. Also, agiven polypeptide may contain more than one type of modification.Blockage of the amino or carboxyl group in a polypeptide, or both, by acovalent modification, is common in naturally-occurring and syntheticpolypeptides. For instance, the amino-terminal residue of polypeptidesmade in E. coli, prior to proteolytic processing, is oftenN-formylmethionine. The modifications can be a function of how theprotein is made. For recombinant polypeptides, for example, themodifications are determined by the host cell posttranslationalmodification capacity and the modification signals in the polypeptideamino acid sequence. Accordingly, when glycosylation is desired, apolypeptide can be expressed in a glycosylating host, generally aeukaryotic cell. Insect cells often carry out the same posttranslationalglycosylations as mammalian cells and, for this reason, insect cellexpression systems have been developed to efficiently express mammalianproteins having native patterns of glycosylation. Similar considerationsapply to other modifications.

Variant polypeptides can be fully functional or can lack function in oneor more activities, e.g., in any of the functions or activitiesdescribed above. Among the many types of useful variations are, e.g.,those that exhibit alteration of receptor activity. For example, oneembodiment involves a variation at the binding site that results inbinding of a ligand, but without receptor activity. A further usefulvariation at the same site can result in altered affinity for aparticular ligand of the receptor. Another useful variation provides afusion protein in which one or more domains or subregions areoperationally fused to one or more domains or subregions from anothernicotinic alpha-7 isoform or family member.

As noted above, the polypeptides of the present invention include, e.g.,isolated polypeptides comprising the sequence of FIG. 2, 4, or 6 (SEQ IDNOS: 2, 4, or 6) (in particular the mature polypeptides) and fragmentsthereof. The polypeptides of the invention also include polypeptides,which have varying degrees of sequence homology (identity) thereto. Theinvention also encompasses polypeptides having a lower degree ofsequence identity, but having sufficient similarity so as to perform oneor more of the functions or activities exhibited by the rhesus monkeynicotinic alpha-7 receptor.

In accordance with the present invention, the term “percent identity” or“percent identical,” when referring to a sequence, means that a sequenceis compared to a claimed or described sequence after alignment of thesequence to be compared (the “Compared Sequence”) with the described orclaimed sequence (the “Reference Sequence”). The Percent Identity isthen determined according to the following formula:Percent Identity=100[1−(C/R)]wherein C is the number of differences between the Reference Sequenceand the Compared Sequence over the length of alignment between theReference Sequence and the Compared Sequence wherein (i) each base oramino acid in the Reference Sequence that does not have a correspondingaligned base or amino acid in the Compared Sequence and (ii) each gap inthe Reference Sequence and (iii) each aligned base or amino acid in theReference Sequence that is different from an aligned base or amino acidin the Compared Sequence, constitutes a difference; and R is the numberof bases or amino acids in the Reference Sequence over the length of thealignment with the Compared Sequence with any gap created in theReference Sequence also being counted as a base or amino acid.

If an alignment exists between the Compared Sequence and the ReferenceSequence for which the percent identity as calculated above is aboutequal to or greater than a specified minimum Percent Identity then theCompared Sequence has the specified minimum percent identity to theReference Sequence even though alignments may exist in which thehereinabove calculated Percent Identity is less than the specifiedPercent Identity.

In a preferred embodiment, the length of a reference sequence alignedfor comparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, or 90% of the length of the referencesequence.

The description herein for percent identity or percent homology isintended to apply equally to nucleotide or amino acid sequences.

The comparison of sequences and determination of percent identity andsimilarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991).

A preferred, non-limiting example of such a mathematical algorithm isdescribed in Karlin et al. (1993) Proc. Natl. Acad. Sci. USA90:5873-5877. Such an algorithm is incorporated into the NBLAST andXBLAST programs (version 2.0) as described in Altschul et al. (1997)Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,NBLASST) can be used. In one embodiment, parameters for sequencecomparison can be set at score=100, wordlength-12, or can be varied(e.g., W=5 or W=20).

In a preferred embodiment, the percent identity between two amino acidsequences is determined using the Needleman et al. (1970) (J. Mol. Biol.48:444-453) algorithm which has been incorporated into the GAP programin the GCG software package using either a BLOSUM 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1,2,3,4,5 or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program I the GCG software package (Devereux et al. (1984)Nucleic Acids Res. 12 (1):387) using a NWSgapdna. CMP matrix and a gapweight of 40, 50, 60, 70, or 80 and a length weight of 1,2,3,4,5 or 6.

Another preferred, non-limiting example of a mathematical algorithmutilized for the comparison of sequences is the algorithm of Myers andMiller, CABIOS (1989). Such an algorithm is incorporated into the ALIGNprogram (version 2.0) which is part of the CGC sequence alignmentsoftware package. When utilizing the ALIGN program for comparing aminoacid sequences, a PAM120 weight residue table, a gap length penalty of12, and a gap penalty of 4 can be used. Additional algorithms forsequence analysis are known in the art and include ADVANCE and ADAM asdescribed in Torellis et al. (1994) Comput. Appl. Biosci. 10:3-5; andFASTA described in Pearson et al. (1988) PNAS 85:2444-8.

In accordance with the present invention, the term “substantiallyhomologous,” when referring to a protein sequence, means that the aminoacid sequences are at least about 90-95%, preferably 97-99% or moreidentical.

Conditions of “high stringency,” as used herein, means, for example,incubating a blot overnight (e.g., at least 12 hours) with a longpolynucleotide probe in a hybridization solution containing, e.g., about5×SSC, 0.5% SDS, 100 μg/ml denatured salmon sperm DNA and 50% formamide,at 42° C. Blots can be washed at high stringency conditions that allow,e.g., for less than 5% bp mismatch (e.g., wash twice in 0.1×SSC and 0.1%SDS for 30 min at 65° C.), thereby selecting sequences having, e.g., 95%or greater sequence identity.

Other non-limiting examples of high stringency conditions include afinal wash at 65° C. in aqueous buffer containing 30 mM NaCl and 0.5%SDS. Another example of high stringent conditions is hybridization in 7%SDS, 0.5 M NaPO₄, pH 7, 1 mM EDTA at 50° C., e.g., overnight, followedby one or more washes with a 1% SDS solution at 42° C. Whereas highstringency washes can allow for less than 5% mismatch, reduced or lowstringency conditions can permit up to 20% nucleotide mismatch.Hybridization at low stringency can be accomplished as above, but usinglower formamide conditions, lower temperatures and/or lower saltconcentrations, as well as longer periods of incubation time.

Polypeptides, and fragments or variants thereof, within the presentinvention may also contain unbroken stretches of amino acids, e.g.,about 6, 8, 10, 12, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80 or 90amino acids. A further preferred fragment excludes the signal sequence(for example, signal sequence may include amino acids 1-22 of FIG. 2, 4,or 6 (SEQ ID NOS: 2, 4, or 6).

As used with respect to the polypeptides (and polynucleotides) of thepresent invention, the term fragment refers to a sequence that is asubset of a larger sequence (i.e., a continuous or unbroken sequence ofresidues within a larger sequence). Peptides already present in the artare, of course, excluded.

The polypeptides, and fragments thereof, of the present invention may befound in the cells and tissues of any species of animal, but arepreferably found in cells from mammals, e.g., mouse, rat, rabbit, farmanimals, pets, etc., especially the cells of primates. In any givenanimal, the polypeptides and fragments thereof within the presentinvention may be found in a variety of tissues. Methods of determiningthe tissue or cellular location of such polypeptides are conventionaland include, e.g., conventional methods of immunohistochemistry. Variousalpha-7 receptors are found in, e.g., many brain regions, hippocampus,thalamus, cerebellum, and hypothalamus. (Quik, M., J. of ComparativeNeurology (2000) 425, 58-69.)

In particular, the alpha-7 receptor polypeptides and fragments thereofof the invention are found in cells, tissues and organs of the nervoussystem, most especially the brain, for example in the various regions ofthe hippocampus.

Nucleic Acids

As discussed above, the invention includes, e.g., cDNA (SEQ ID NOs: 1 or3) encoding full length polypeptides of the invention, and fragmentsthereof.

The polynucleotides of SEQ FIG. 1, 3, or 5 (SEQ ID NOS: 1, 3, or 5)contain open reading frames available for the coding of polypeptideamino acid sequences. For the sequence of SEQ ID NO: 1, the open readingframe (or ORF) coding for the polypeptide of SEQ ID NO: 2 is found atnucleotides 7-1515 (with nucleotides 1513-1515 representing the “TAA”termination codon). For the sequence of SEQ ID NO: 3, the open readingframe (or ORF) coding for the polypeptide of SEQ ID NO. 4 is found atnucleotides 7-1515 (with nucleotides 1513-1515 representing the “TAA”termination codon). For the sequence of SEQ ID NO: 5, the open readingframe (or ORF) coding for the polypeptide of SEQ ID NO: 6 is found atnucleotide 7-1515 with nucleotides 1513-1515 representing the “TAA”termination codon).

As used herein, the phrase “an isolated polynucleotide which is SEQ IDNO,” or “an isolated polynucleotide which is selected from SEQ ID NO,”refers to an isolated nucleic acid molecule from which the recitedsequence was obtained (i.e., the mRNA). Because of sequencing errors,typographical errors, etc., the actual naturally-occurring sequence maydiffer from a SEQ ID listed herein. Thus, the phrase indicates thespecific molecule from which the sequence was derived, rather than amolecule having that exact recited nucleotide sequence, analogously tohow a culture depository number refers to a specific cloned fragment ina cryotube.

A polynucleotide of the present invention may be a recombinantpolynucleotide, a natural polynucleotide, or a synthetic orsemi-synthetic polynucleotide, or combinations thereof. As used herein,the terms polynucleotide, oligonucleotide, oligomer and nucleic acid areinterchangeable.

As used herein, the term “gene” means a segment of DNA involved inproducing a polypeptide chain; it may include regions preceding andfollowing the coding region (leader and trailer) as well as interveningsequences (introns) between individual coding segments (exons). cDNAslack the corresponding introns. The invention includes isolated genes(e.g., genomic clones) which encode polypeptides of the invention.

Polynucleotides of the invention may be RNA, PNA, or DNA, e.g., cDNA,genomic DNA, and synthetic or semi-synthetic DNA, or combinationsthereof. The DNA may be triplex, double-stranded or single-stranded, andif single stranded, may be the coding strand or non-coding (anti-sense)strand. It can comprise hairpins or other secondary structures. The RNAincludes oligomers (including those having sense or antisense strands),mRNAs (e.g., having the alternative splices of the alpha-7 receptor),polyadenylated RNA, total RNA, single strand or double strand RNA, orthe like. DNA/RNA duplexes are also encompassed by the invention.

The polynucleotides and fragments thereof of the present invention maybe of any size that is compatible with the objects of the invention,e.g., of any desired size that is effective to achieve a desiredspecificity when used as a probe. Polynucleotides may range in size,e.g., from the smallest specific probe (e.g., about 10-12 nucleotides )to greater than a full-length cDNA, e.g., in the case of a fusionpolynucleotide or a polynucleotide that is part of a genomic sequence;fragments may be as large as, e.g., one nucleotide shorter than afull-length cDNA.

A fragment of a polynucleotide according to the invention may be used,e.g., as a hybridization probe, as discussed elsewhere herein.

Many types of variants of polynucleotides are encompassed by theinvention including, e.g., (i) one in which one or more of thenucleotides is substituted with another nucleotide, or which isotherwise mutated; or (ii) one in which one or more of the nucleotidesis modified, e.g., includes a subtituent group; or (iii) one in whichthe polynucleotide is fused with another compound, such as a compound toincrease the half-life of the polynucleotide; or (iv) one in whichadditional nucleotides are covalently bound to the polynucleotide, sucha sequences encoding a leader or secretory sequence or a sequence whichis employed for purification of the polypeptide. The additionalnucleotides may be from a heterologous source, or may be endogenous tothe natural gene.

Polynucleotide variants belonging to type (i) above include, e.g.,polymorphisms, including single nucleotide polymorphisms (SNPs), andmutants. Variant polynucleotides can comprise, e.g., one or moreadditions, insertions, deletions, substitutions, transitions,transversions, inversions, chromosomal translocations, variantsresulting from alternative splicing events, or the like, or anycombinations thereof

A coding sequence which encodes a polypeptide (e.g., a maturepolypeptide) of the invention may be identical to the coding sequenceshown in FIG. 1, 3, or 5 (SEQ ID NOS: 1, 3, or 5) or a fragment thereof,or may be a different coding sequence, which coding sequence, as aresult of the redundancy or degeneracy of the genetic code, encodes thesame polypeptide as the DNA of FIG. 1, 3, or 5 (SEQ ID NOS: 1, 3, or 5)or a fragment thereof Such a peptide is sometimes referred to herein asa “degenerate variant.” Alternatively, the coding sequence may encode apolypeptide that is substantially homologous to the polypeptide of FIG.2, 4, or 6 (SEQ ID NOS: 2, 4, or 6) or a fragment thereof

A polynucleotide of the invention may have a coding sequence which is anaturally or non-naturally occurring allelic variant of a codingsequence encompassed by the sequence in SEQ ID NO: 1. As known in theart, an allelic variant is an alternate form of a polynucleotidesequence which may have a substitution, deletion or addition of one ormore nucleotides, which in general does not substantially alter thefunction of the encoded polypeptide.

Other variant sequences, located in a coding sequence or in a regulatorysequence, may affect (enhance or decrease) the production of, or thefunction or activity of, a polypeptide of the invention.

Polynucleotide variants belonging to type (ii) above include, e.g.,modifications such as the attachment of detectable markers (avidin,biotin, radioactive elements, fluorescent tags and dyes, energy transferlabels, energy-emitting labels, binding partners, etc.) or moietieswhich improve expression, uptake, cataloging, tagging, hybridization,detection, and/or stability. The polynucleotides can also be attached tosolid supports, e.g., nitrocellulose, magnetic or paramagneticmicrospheres (e.g., as described in U.S. Pat. Nos. 5,411,863; 5,543,289;for instance, comprising ferromagnetic, supermagnetic, paramagnetic,superparamagnetic, iron oxide and polysaccharide), nylon, agarose,diazotized cellulose, latex solid microspheres, polyacrylamides, etc.,according to a desired method. See, e.g., U.S. Pat. Nos. 5,470,967;5,476,925; 5,478,893.

Polynucleotide variants belonging to type (iii) above are well known inthe art and include, e.g., various lengths of polyA⁺ tail, 5′ capstructures, and nucleotide analogs, e.g., inosine, thionucleotides, orthe like.

Polynucleotide variants belonging to type (iv) above include, e.g., avariety of chimeric, hybrid or fusion polynucleotides. For example. apolynucleotide of the invention can comprise a coding sequence andadditional non-naturally occurring or heterologous coding sequence(e.g., sequences coding for leader, signal, secretory, targeting,enzymatic, fluorescent, antibiotic resistance, and other functional ordiagnostic peptides); or a coding sequence and non-coding sequences,e.g., untranslated sequences at either a 5′ or 3′ end, or dispersed inthe coding sequence, e.g., introns.

More specifically, the present invention includes polynucleotideswherein the coding sequence for the polypeptide (e.g., a maturepolypeptide) is fused in the same reading frame to a polynucleotidesequence (e.g., a heterologous sequence), e.g. one which aids inexpression and secretion of a polypeptide from a host cell, for example,a leader sequence which functions as a secretory sequence forcontrolling transport of a polypeptide from the cell and/or atransmembrane anchor which facilitates attachment of the polypeptide toa cellular membrane. A polypeptide having a leader sequence is apreprotein and may have the leader sequence cleaved by the host cell toform a mature form of the polypeptide. The polynucleotides may alsoencode for a proprotein which is the mature protein plus additionalN-terminal amino acid residues. A mature protein having a prosequence isa proprotein and is generally an inactive form of the protein. Once theprosequence is cleaved an active protein remains.

Polynucleotides of the present invention may also have a coding sequencefused in frame to a marker sequence that allows for identificationand/or purification of the polypeptide of the present invention. Themarker sequence may be, e.g., a hexa-histidine tag (e.g., as supplied bya pQE-9 vector) to provide for purification of the mature polypeptidefused to the marker in the case of a bacterial host, or, for example,the marker sequence may be a hemagglutinin (HA) tag when a mammalianhost, e.g. COS-7 cells, is used. The HA tag corresponds to an epitopederived from the influenza hemagglutinin protein (Wilson, I., et al.,Cell, 37:767 (1984)).

Other types of polynucleotide variants will be evident to one of skillin the art. For example, the nucleotides of a polynucleotide can bejoined via various known linkages, e.g., ester, sulfamate, sulfamide,phosphorothioate, phosphoramidate, methylphosphonate, carbamate, etc.,depending on the desired purpose, e.g., resistance to nucleases, such asRNAse H, improved in vivo stability, etc. See, e.g., U.S. Pat. No.5,378,825. Any desired nucleotide or nucleotide analog can beincorporated, e.g., 6-mercaptoguanine, 8-oxo-guanine, etc. Also,polynucleotides of the invention may have a coding sequence derived fromanother genetic locus of an organism, providing it has a substantialhomology to, e.g., part or all of the sequence of FIG. 1, 3, or 5 (SEQID NOS: 1, 3, or 5) or from another organism (e.g., an ortholog). It isunderstood that variants exclude any sequences disclosed prior to theinvention.

Polynucleotides according to the present invention can be labeledaccording to any desired method. The polynucleotide can be labeled usingradioactive tracers such as, e.g., ³²P, ³⁵S, ³H, or ¹⁴C. The radioactivelabeling can be carried out according to any method, such as, forexample, terminal labeling at the 3′ or 5′ end using a radiolabelednucleotide, polynucleotide kinase (with or without dephosphorylationwith a phosphatase) or a ligase (depending on the end to be labeled). Anon-radioactive labeling can also be used, combining a polynucleotide ofthe present invention with residues having immunological properties(antigens, haptens), a specific affinity for certain reagents (ligands),properties enabling detectable enzyme reactions to be completed (enzymesor coenzymes, enzyme substrates, or other substances involved in anenzymatic reaction), or characteristic physical properties, such asfluorescence or the emission or absorption of light at a desiredwavelength, etc.

The present invention includes polynucleotides encoding all of thepolypeptides and fragments or variants thereof, as disclosedhereinabove. For example, a polynucleotide of the invention may comprisea sequence which has a sequence identity of at least about 65-100%,(e.g., at least about 70-75%, 80-85%, 90-95% or 97-99%) to, or which issubstantially homologous to, or which hybridizes under conditions ofhigh stringency to, the nucleotide sequence of SEQ ID NO: 1, or to afragment thereof; or which is complementary to one of those sequences.

The term “substantially homologous,” when referring to polynucleotidesequences, means that the nucleotide sequences are at least about 90-95%or 97-99% or more identical.

Constructs

The present invention also relates to recombinant constructs thatcontain vectors plus polynucleotides of the present invention. Suchconstructs comprise a vector, such as a plasmid or viral vector, intowhich a polynucleotide sequence of the invention has been inserted, in aforward or reverse orientation.

Large numbers of suitable vectors are known to those of skill in theart, and many are commercially available. The following vectors areprovided by way of example; Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pBS, pD10, phagescript, psiX174, pBluescript SK, pBSKS, pNH8A, pNH16a,pNH18A, pNH46A (Stratagene); pTRC99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia); Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid orvector may be used as long as it is replicable and viable in the host.

In a preferred embodiment, the vector is an expression vector, intowhich a polynucleotide sequence of the invention is inserted so as to beoperatively linked to an appropriate expression control (regulatory)sequence(s) (e.g., promoters and/or enhancers) which directs mRNAsynthesis. Appropriate expression control sequences, e.g., regulatablepromoter or regulatory sequences known to control expression of genes inprokaryotic or eukaryotic cells or their viruses, can be selected forexpression in prokaryotes (e.g., bacteria), yeast, plants, mammaliancells or other cells. Preferred expression control sequences are derivedfrom highly-expressed genes, e.g., from operons encoding glycolyticenzymes such as 3-phosphoglycerate kinase (PGK), α-factor, acidphosphatase, or heat shock proteins, among others. Such expressioncontrol sequences can be selected from any desired gene, e.g using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors for such selection are pKK232-8 andpCM7.

Particular named bacterial promoters which can be used include lacI,lacZ, T3, T7, gpt, lambda P_(R), P_(L) and trp. Eukaryotic promotersinclude CMV immediate early, HSV thymidine kinase, early and late SV40,adenovirus promoters, LTRs from retrovirus, and mouse metallothionein-I.Selection of the appropriate vector and promoter is well within thelevel of ordinary skill in the art.

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes can be increased by inserting an enhancersequence into the expression vector. Enhancers are cis-acting elementsof DNA, usually about from 10 to 300 bp that act on a promoter toincrease its transcription. Representative examples include the SV40enhancer on the late side of the replication origin bp 100 to 270, acytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers.

Generally, recombinant expression vectors also include origins ofreplication. An expression vector may contain a ribosome binding sitefor translation initiation, a transcription termination sequence, apolyadenylation site, splice donor and acceptor sites, and/or 5′flanking or non-transcribed sequences. DNA sequences derived from theSV40 splice and polyadenylation sites may be used to provide requirednontranscribed genetic elements. The vector may also include appropriatesequences for amplifying expression. In addition, expression vectorspreferably contain one or more selectable marker genes to provide aphenotypic trait for selection of transformed host cells such asdihydrofolate reductase or neomycin resistance for eukaryotic cellculture, or such as tetracycline or ampicillin resistance in E. coli.

Large numbers of suitable expression vectors are known to those of skillin the art, and many are commercially available. Suitable vectorsinclude chromosomal, nonchromosomal and synthetic DNA sequences, e.g.,derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeastplasmids; vectors derived from combinations of plasmids and phage DNA,viral DNA such as vaccinia, adenovirus, adeno-associated virus, TMV,fowl pox virus, and pseudorabies. However, any other vector may be usedas long as it is replicable and viable in a host. Appropriate cloningand expression vectors for use with prokaryotic and eukaryotic hosts aredescribed, e.g., by Sambrook, et al., Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor, N.Y., (1989), Wu et al,Methods in Gene Biotechnology (CRC Press, New York, N.Y., 1997),Recombinant Gene Expression Protocols, in Methods in Molecular Biology,Vol. 62, (Tuan, ed., Humana Press, Totowa, N.J., 1997), and CurrentProtocols in Molecular Biology, (Ausabel et al, Eds.,), John Wiley &Sons, NY (1994-1999).

In a preferred embodiment, a Baculovirus-based expression system isused. Baculoviruses represent a large family of DNA viruses that infectmostly insects. The prototype is the nuclear polyhedrosis virus (AcMNPV)from Autographa californica, which infects a number of lepidopteranspecies. One advantage of the baculovirus system is that recombinantbaculoviruses can be produced in vivo. Following co-transfection withtransfer plasmid, most progeny tend to be wild type and a good deal ofthe subsequent processing involves screening. To help identify plaques,special systems are available that utilize deletion mutants. By way ofnon-limiting example, a recombinant AcMNPV derivative (called BacPAK6)has been reported in the literature that includes target sites for therestriction nuclease Bsu36I upstream of the polyhedrin gene (and withinORF 1629) that encodes a capsid gene (essential for virus viability).Bsf36I does not cut elsewhere in the genome and digestion of the BacPAK6deletes a portion of the ORF1629, thereby rendering the virusnon-viable. Thus, with a protocol involving a system like Bsu361-cutBacPAK6 DNA most of the progeny are non-viable so that the only progenyobtained after co-transfection of transfer plasmid and digested BacPAK6is the recombinant because the transfer plasmid, containing theexogenous DNA, is inserted at the Bsu36I site thereby rendering therecombinants resistant to the enzyme. See Kitts and Possee, A method forproducing baculovirus expression vectors at high frequency,BioTechniques, 14, 810-817 (1993). For general procedures, see King andPossee, The Baculovirus Expression System: A Laboratory Guide, Chapmanand Hall, New York (1992) and Recombinant Gene Expression Protocols, inMethods in Molecular Biology, Vol. 62, (Tuan, ed., Humana Press, Totowa,N.J., 1997), at Chapter 19, pp. 235-246.

Appropriate DNA sequences may be inserted into a vector by any of avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art. Conventional procedures for this and othermolecular biology techniques discussed herein are found in many readilyavailable sources, e.g., Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989). Ifdesired, a heterologous structural sequence is assembled in anexpression vector in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium.

Transformed Cells and Methods of Producing Polypeptides of the Invention

The present invention also relates to host cells which aretransformed/transfected/transduced (i.e, wherein a polynucleotide of thepresent invention has been introduced into the host cell, and istherefore not the endogenous, naturally-occurring gene which is normallypresent in the cell) with constructs such as those described above, andto progeny of said cells, especially where such cells result in a stablecell line that can be used for assays of nicotinic alpha-7 receptoractivity, e.g., in order to identify agents which modulate nicotinicalpha-7 receptor activity, and/or for production (e.g., preparativeproduction) of the polypeptides of the invention. The host cell cancontain a vector comprising a polynucleotide operably linked to anexpression control sequence (e.g., a promoter) which enables expressionof the polypeptide. Once expressed in the cell, the alpha-7 nicotinicpolypeptides can assemble with each other to form a functionalhomo-pentameric channel which is located on the surface of the cell(i.e., on the surface cell membrane). As representative examples ofappropriate hosts, there may be mentioned: bacterial cells, such as E.coli, Streptomyces, Salmonella typhimurium; fungal cells, such as yeast;insect cells such as Drosophila S2 and Spodoptera Sf9 (and other insectexpression systems); animal cells, including mammalian cells such asCHO, COS (e.g., the COS-7 lines of monkey kidney fibroblasts describedby Gluzman, Cell, 23:175 (1981)), C127, 3T3, CHO, HeLa, BHK or Bowesmelanoma cell lines; muscle cells; neuronal cells; brain cells; oocytes(such as from Xenopus or other frog species), QT6 (ATCC CRL-1708), QM7(ATCC CRL-1962), plant cells, etc. The selection of an appropriate hostis deemed to be within the knowledge of those skilled in the art basedon the teachings herein.

In another embodiment, the host cells are insect cells of Spodopteraspecies, most especially SF9 cells, from Spodoptera frugiperda.Polypeptides (e.g., full length polypeptides) of the present inventionare readily obtainable from insect cells using a baculovirus expressionvector. Such expression is readily characterized using methods wellknown in the art.

Introduction of a construct into a host cell can be effected by, e.g.,calcium phosphate transfection, DEAE-Dextran mediated transfection,lipofection a gene gun, or electroporation (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)).

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter can beinduced by appropriate means (e.g., temperature shift or chemicalinduction) if desired, and cells cultured for an additional period. Theengineered host cells are cultured in conventional nutrient mediamodified as appropriate for activating promoters (if desired), selectingtransformants or amplifying the genes of the present invention. Theculture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification. Alternatively, when a heterologous polypeptide is secretedfrom the host cell into the culture fluid, supernatants of the culturefluid can be used as a source of the protein. Microbial cells employedin expression of proteins can be disrupted by any convenient method,including freeze-thaw cycling, sonication, mechanical disruption, or useof cell lysing agents, such methods being well known to those skilled inthe art.

The polypeptide can be recovered and purified from recombinant cellcultures by conventional methods including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography, or the like. Protein refolding steps can be used, asnecessary, in completing configuration of the mature protein. Highperformance liquid chromatography (HPLC) can be employed for finalpurification steps.

In addition to the methods described above for producing polypeptidesrecombinantly from a prokaryotic or eukaryotic host, polypeptides of theinvention can be prepared from natural sources, or can be prepared bychemical synthetic procedures (e.g., synthetic or semi-synthetic), e.g.,with conventional peptide synthesizers. Cell-free translation systemscan also be employed to produce such proteins using RNAs derived fromthe DNA constructs of the present invention. Proteins of the inventioncan also be expressed in, and isolated and/or purified from, transgenicanimals or plants. Procedures to make and use such transgenic organismsare conventional in the art. Some such procedures are describedelsewhere herein.

Antibodies, Antigen-binding Fragments or Other Specific Binding Partners

The polypeptides, their fragments or variants thereof, or cellsexpressing them can also be used as immunogens to produce specificantibodies, or antigen-binding fragments, thereto. By a “specific”antibody or antigen-binding fragment is meant one which bindsselectively (preferentially) to nicotinic alpha-7 receptor of theinvention, or to a fragment or variant thereof. An antibody “specific”for a polypeptide means that the antibody recognizes a defined sequenceof amino acids within or including the polypeptide. Preferred antibodiesare those that recognize epitopes which are specific for the monkeyalpha-7 nicotinic acetylcholine receptor of the present invention, e.g.,regions which differ in sequence identity between the correspondingprotein expressed in other species.

Antibodies of the invention can be, for example, polyclonal ormonoclonal antibodies. The present invention also includes chimeric,recombinant, single chain, and partially or fully humanized antibodies,as well as Fab fragments, or the product of a Fab expression library,and fragments thereof. The antibodies can be IgM, IgG, subtypes, IgG2A,JgG1, etc. Various procedures known in the art may be used for theproduction of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained, e.g., by directinjection of the polypeptides into an animal or by administering thepolypeptides to an animal, e.g., goat, rabbit, mouse, chicken, etc.,preferably a non-human. The antibody so obtained will then bind thepolypeptide itself. In this manner, even a sequence encoding only afragment of the polypeptides can be used to generate antibodies bindingthe whole native polypeptides. Such antibodies can then be used toisolate the polypeptide from tissue expressing that polypeptide.Antibodies can also be generated by administering naked DNA. See, e.g.,U.S. Pat. Nos. 5,703,055; 5,589,466; and 5,580,859.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include, e.g., the hybridoma technique (Kohler and Milstein,1975, Nature, 256:495-497), the trioma technique, the human B-cellhybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), andthe EBV-hybridoma technique to produce human monoclonal antibodies(Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, Inc., pp. 77-96).

Techniques described for the production of single chain antibodies(e.g., U.S. Pat. No. 4,946,778) can be adapted to produce single chainantibodies to immunogenic polypeptide products of this invention. Also,transgenic animals may be used to express partially or fully humanizedantibodies to immunogenic polypeptide products of this invention.

The invention also relates to other specific binding partners, whichinclude, e.g., aptamers and PNA.

Transgenic and Knockout Animals

The invention disclosed herein also relates to a non-human transgenicanimal comprising within its genome one or more copies of thepolynucleotides encoding the novel polypeptides of the invention. Thetransgenic animals of the invention may contain within their genomemultiple copies of the polynucleotides encoding the polypeptides of theinvention, or one copy of a gene encoding such polypeptide but whereinsaid gene is linked to a promoter (e.g., a regulatable promoter) thatwill direct expression (preferably overexpression) of said polypeptidewithin some, or all, of the cells of said transgenic animal. In apreferred embodiment, expression of a polypeptide of the inventionoccurs preferentially in brain tissue, e.g., hippocampus. A variety ofnon-human transgenic organisms are encompassed by the invention,including e.g., drosophila, C.elegans, zebrafish and yeast. Thetransgenic animal of the invention is preferably a mammal, e.g., a cow,goat, sheep, rabbit, non-human primate, monkey, rhesus monkey, rat, ormouse.

Methods of producing transgenic animals are well within the skill ofthose in the art, and include, e.g., homologous recombination,mutagenesis (e.g., ENU, Rathkolb et al., Exp. Physiol., 85(6):635-644,2000), and the tetracycline-regulated gene expression system (e.g., U.S.Pat. No. 6,242,667), and will not be described in detail herein. [Seee.g., Wu et al, Methods in Gene Biotechnology, CRC 1997, pp. 339-366;Jacenko, O., Strategies in Generating Transgenic Animals, in RecombinantGene Expression Protocols, Vol. 62 of Methods in Molecular Biology,Humana Press, 1997, pp 399-424]

Transgenic organisms are useful, e.g. for providing a source of apolynucleotide or polypeptide of the invention, or for identifyingand/or characterizing agents that modulate expression and/or activity ofsuch a polynucleotide or polypeptide. Transgenic animals are also usefulas models for disease conditions related to, e.g., overexpression of apolynucleotide or polypeptide of the invention.

The present invention also relates to a non-human knockout animal whosegenome lacks or fails to express a functional nicotinic alpha-7 receptoror functional analog thereof (i.e., the gene is functionally disrupted),such animal commonly being referred to as a “knockout” animal,especially a “knock-out mouse.”

Functional disruption of the gene can be accomplished in any effectiveway, including, e.g., introduction of a stop codon into any part of thecoding sequence such that the resulting polypeptide is biologicallyinactive (e.g., because it lacks a catalytic domain, a ligand bindingdomain, etc.), introduction of a mutation into a promoter or otherregulatory sequence that is effective to turn it off, or reducetranscription of the gene, insertion of an exogenous sequence into thegene which inactivates it (e.g., which disrupts the production of abiologically-active polypeptide or which disrupts the promoter or othertranscriptional machinery), deletion of sequences from the nicotinicalpha-7 receptor gene, etc. Examples of transgenic animals havingfunctionally disrupted genes are well known, e.g., as described in U.S.Pat. Nos. 6,239,326, 6,225,525, 6,207,878, 6,194,633, 6,187,992,6,180,849, 6,177,610, 6,100,445, 6,087,555, 6,080,910, 6,069,297,6,060,642, 6,028,244, 6,013,858, 5,981,830, 5,866,760, 5,859,314,5,850,004, 5,817,912, 5,789,654, 5,777,195, and 5,569,824. Knock-outscan be homozygous or heterozygous.

For creating functional disrupted genes, and other gene mutations,homologous recombination technology is of special interest since itallows specific regions of the genome to be targeted. Using homologousrecombination methods, genes can be specifically inactivated, specificmutations can be introduced, and exogenous sequences can be introducedat specific sites. These methods are well known in the art, e.g., asdescribed in the patents above. See, also, Robertson, Biol. Reproduc.,44(2):238-245, 1991. Generally, the genetic engineering is performed inan embryonic stem (ES) cell, or other pluripotent cell line (e.g., adultstem cells, EG cells), and that genetically-modified cell (or nucleus)is used to create a whole organism. Nuclear transfer can be used incombination with homologous recombination technologies.

For example, nicotinic alpha-7 locus can be disrupted in monkey ES cellsusing a positive-negative selection method (e.g., Mansour et al.,Nature, 336:348-352, 1988). In this method, a targeting vector can beconstructed which comprises a part of the gene to be targeted. Aselectable marker, such as neomycin resistance genes, can be insertedinto an alpha-7 exon present in the targeting vector, disrupting it.When the vector recombines with the ES cell genome, it disrupts thefunction of the gene. The presence in the cell of the vector can bedetermined by expression of neomycin resistance. See, e.g., U.S. Pat.No. 6,239,326. Cells having at least one functionally disrupted gene canbe used to make chimeric and germline animals, e.g., animals havingsomatic and/or germ cells comprising the engineered gene. Homozygousknock-out animals can be obtained from breeding heterozygous knock-outanimals. See, e.g., U.S. Pat. No. 6,225,525.

The present invention also relates to a transgenic non-human animalwhose genome comprises one or more genes coding for monkey alpha-7receptor disclosed herein in place of the mammalian gene otherwisecoding for said non-human isoform.

A knock-out animal, or animal cell, lacking one or more fictionalnicotinic alpha-7 receptor genes can be useful in a variety ofapplications, including as an animal model for an nicotinic alpha-7receptor-mediated or related condition, for drug screening assays, as asource of tissues deficient in nicotinic alpha-7 receptor activity, asthe starting material for generating an animal in which the endogenousnicotinic alpha-7 receptor is replaced with monkey alpha-7 receptor, andany of the utilities mentioned in any issued U.S. Patent on transgenicanimals, including, U.S. Pat. Nos. 6,239,326, 6,225,525, 6,207,878,6,194,633, 6,187,992, 6,180,849, 6,177,610, 6,100,445, 6,087,555,6,080,910, 6,069,297, 6,060,642, 6,028,244, 6,013,858, 5,981,830,5,866,760, 5,859,314, 5,850,004, 5,817,912, 5,789,654, 5,777,195, and5,569,824. For instance, nicotinic alpha-7 receptor deficient animalcells can be utilized to study activities related to, e.g., neuronaldiseases, such as Alzheimer's disease and schizophrenia. By knocking-outnicotinic alpha-7 receptors e.g., one at a time, the physiologicalpathways using nicotinic alpha-7 receptors can be dissected out andidentified.

In addition to the methods mentioned above, transgenic or knock-outanimals can be prepared according to known methods, including, e.g., bypronuclear injection of recombinant genes into pronuclei of 1-cellembryos, incorporating an artificial yeast chromosome into embryonicstem cells, gene targeting methods, embryonic stem cell methodology,cloning methods, nuclear transfer methods. See, also, e.g., U.S. Pat.Nos. 4,736,866; 4,873,191; 4,873,316; 5,082,779; 5,304,489; 5,174,986;5,175,384; 5,175,385; 5,221,778; Gordon et al., Proc. Natl. Acad. Sci.,77:7380-7384, 1980; Palmiter et al., Cell, 41:343-345, 1985; Palmiter etal., Ann. Rev. Genet., 20:465-499, 1986; Askew et al., Mol. Cell. Bio.,13:4115-4124, 1993; Games et al. Nature, 373:523-527, 1995; Valanciusand Smithies, Mol. Cell. Bio., 11:1402-1408, 1991; Stacey et al., Mol.Cell. Bio., 14:1009-1016, 1994; Hasty et al., Nature, 350:243-246, 1995;Rubinstein et al., Nucl. Acid Res., 21:2613-2617,1993; Cibelli et al.,Science, 280:1256-1258, 1998. For guidance on recombinase excisionsystems, see, e.g., U.S. Pat. Nos. 5,626,159, 5,527,695, and 5,434,066.See also, Orban, P. C., et al., “Tissue- and Site-Specific DNARecombination in Transgenic Mice,” Proc. Natl. Acad. Sci. USA,89:6861-6865 (1992); O'Gorman, S., et al., “Recombinase-Mediated GeneActivation and Site-Specific Integration in Mammalian Cells,” Science,251:1351-1355 (1991); Sauer, B., et al., “Cre-stimulated recombinationat loxP-Containing DNA sequences placed into the mammalian genome,”Polynucleotides Research, 17(1):147-161 (1989); Gagneten, S. et al.(1997) Nucl. Acids Res. 25:3326-3331; Xiao and Weaver (1997) Nucl. AcidsRes. 25:2985-2991; Agah, R. et al. (1997) J. Clin. Invest. 100:169-179;Barlow, C. et al. (1997) Nucl. Acids Res. 25:2543-2545; Araki, K. et al.(1997) Nucl. Acids Res. 25:868-872; Mortensen, R. N. et al. (1992) Mol.Cell. Biol. 12:2391-2395 (G418 escalation method); Lakhlani, P. P. etal. (1997) Proc. Natl. Acad. Sci. USA 94:9950-9955 (“hit and run”);Westphal and Leder (1997) Curr. Biol. 7:530-533 (transposon-generated“knock-out” and “knock-in”); Templeton, N. S. et al. (1997) Gene Ther.4:700-709 (methods for efficient gene targeting, allowing for a highfrequency of homologous recombination events, e.g., without selectablemarkers); PCT International Publication WO 93/22443(functionally-disrupted).

A polynucleotide according to the present invention can be introducedinto any non-human animal, including a non-human mammal, mouse (Hogan etal., Manipulating the Mouse Embryo: A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1986), pig (Hammer et al.,Nature, 315:343-345, 1985), sheep (Hammer et al., Nature, 315:343-345,1985), cattle, rat, or primate. See also, e.g., Church, 1987, Trends inBiotech. 5:13-19; Clark et al., Trends in Biotech. 5:20-24, 1987); andDePamphilis et al., BioTechniques, 6:662-680, 1988. Transgenic animalscan be produced by the methods described in U.S. Pat. No. 5,994,618, andutilized for any of the utilities described therein.

Conditions Related to Nicotinic Alpha-7 Receptor Expression

The alpha-7 receptor of the instant invention is involved in a varietyof functions and activities, e.g. as discussed elsewhere hereinabove,and aberrant expression and/or activity of nicotinic alpha-7 receptor isassociated with a variety of disease conditions. This invention relates,e.g., to the detection (e.g., determination of the presence or absence)and/or quantitation of polypeptides or polynucleotides of the inventionthat are related to such conditions; and to the diagnosis and/orprevention, treatment, or amelioration of symptoms, of such alpha-7receptor-mediated or nicotinic alpha-7 receptor-related conditions. Theinvention also relates to methods of identifying agents that modulate(i.e., increase or decrease) the expression and/or activity ofpolypeptides or polynucleotides associated with such conditions, and tomethods of identifying polypeptide or polynucleotide alterations ormutants that are associated with such conditions.

Methods of the invention relate to the treatment and/or prophylaxis ofvarious diseases and conditions, particularly psychotic diseases,neurodegenerative diseases involving a dysfunction of the cholinergicsystem, and conditions of memory and/or cognition impairment, including,for example, schizophrenia, anxiety, mania, depression, manic depression[examples of psychotic disorders], Tourette's syndrome, Parkinson'sdisease, Huntington's disease [examples of neurodegenerative diseases],cognitive disorders (such as Alzheimer's disease, Lewy Body Dementia,Amyotrophic Lateral Sclerosis, memory impairment, memory loss, cognitiondeficit, attention deficit, Attention Deficit Hyperactivity Disorder),and other uses such as treatment of nicotine addiction, inducing smokingcessation, treating pain (i.e., analgesic use), providingneuroprotection, and treating jetlag. See, e.g., WO 97/30998; WO99/03850; WO 00/42044; WO 01/36417; Holladay et al., J.Med. Chem.,40:26, 4169-94 (1997); Schmitt et al., Annual Reports Med. Chem.,Chapter 5, 41-51 (2000); Stevens et al., Psychopharmatology, (1998) 136:320-27 (1998); and Shytle et al., Molecular Psychiatry, (2002), 7, pp.525-535.

In one embodiment, methods of the invention relate to psychoticdiseases, neurodegenerative diseases involving a dysfunction of thecholinergic system, and conditions of memory and/or cognitionimpairment, including, for example, schizophrenia, anxiety, mania,depression, manic depression [examples of psychotic disorders],Tourette's syndrome, Parkinson's disease, Huntington's disease [examplesof neurodegenerative diseases], and/or cognitive disorders (such asAlzheimer's disease, Lewy Body Dementia, Amyotrophic Lateral Sclerosis,memory impairment, memory loss, cognition deficit, attention deficit,and Attention Deficit Hyperactivity Disorder).

Neurodegenerative disorders included within the methods of the presentinvention include, but are not limited to, treatment and/or prophylaxisof Alzheimer's diseases, Pick's disease, diffuse Lewy Body disease,progressive supranuclear palsy (Steel-Richardson syndrome), multisystemdegeneration (Shy-Drager syndrome), motor neuron diseases includingamyotrophic lateral sclerosis, degenerative ataxias, cortical basaldegeneration, ALS-Parkinson's-Dementia complex of Guam, subacutesclerosing panencephalitis, Huntington's disease, Parkinson's disease,synucleinopathies, primary progressive aphasia, striatonigraldegeneration, Machado-Joseph disease/spinocerebellar ataxia type 3,olivopontocerebellar degenerations, Gilles De La Tourette's disease,bulbar, pseudobulbar palsy, spinal muscular atrophy, spinobulbarmuscular atrophy (Kennedy's disease), primary lateral sclerosis,familial spastic paraplegia, Werdnig-Hoffmann disease,Kugelberg-Welander disease, Tay-Sach's disease, Sandhoff disease,familial spastic disease, Wohlfart-Kugelberg-Welander disease, spasticparaparesis, progressive multifocal leukoencephalopathy, prion diseases(such as Creutzfeldt-Jakob, Gerstmann-Sträussler-Scheinker disease, Kuruand fatal familial insomnia), and neurodegenerative disorders resultingfrom cerebral ischemia or infarction including embolic occlusion andthrombotic occlusion as well as intracranial hemorrhage of any type(including, but not limited to, epidural, subdural, subarachnoid andintracerebral), and intracranial and intravertebral lesions (including,but not limited to, contusion, penetration, shear, compression andlaceration).

The present invention further relates to memory impairment due to, forexample, mild cognitive impairment due to aging, Alzheimer's disease,schizophrenia, Parkinson's disease, Huntington's disease, Pick'sdisease, Creutzfeld-Jakob disease, depression, aging, head trauma,stroke, CNS hypoxia, cerebral senility, multiinfaret dementia and otherneurological conditions, as well as HIV and cardiovascular diseases.

In addition, the methods of the invention relate to age-related dementiaand other dementias and conditions with memory loss includingage-related memory loss, senility, vascular dementia, diffuse whitematter disease (Binswanger's disease), dementia of endocrine ormetabolic origin, dementia of head trauma and diffuse brain damage,dementia pugilistica and frontal lobe dementia. See, e.g., WO 99/62505.

Amyloid precursor protein (APP) and Aβ peptides derived therefrom, e.g.,Aβ₁₋₄₀, Aβ₁₋₄₂, and other fragments, are known to be involved in thepathology of Alzhemier's disease. The Aβ₁₋₄₂ peptides are not onlyimplicated in neurotoxicity but also are known to inhibit cholinergictransmitter function. Further, it has been determined that Aβ peptidesbind to α-7 nAChRs. Thus, agents which block the binding of the Aβpeptides to α-7 nAChRs are useful for treating neurodegenerativediseases. See, e.g., WO 99/62505. In addition, stimulation α-7 nAChRscan protect neurons against cytotoxicity associated with Aβ peptides.See, e.g., Kihara, T. et al., Ann. Neurol., 1997, 42, 159.

Thus, in accordance with an embodiment of the invention, the methods arerelated to treatment or prevention of dementia in an Alzheimer'spatient, which comprises administering to the subject a therapeuticallyeffective amount of a compound able to inhibit the binding of an amyloidbeta peptide (preferably, Aβ₁₋₄₂) with nAChRs, preferable α-7 nAChRs.

The present invention also provides methods related to treating otheramyloidosis diseases, for example, hereditary cerebral angiopathy,nonneuropathic hereditary amyloid, Down's syndrome, macroglobulinemia,secondary familial Mediterranean fever, Muckle-Wells syndrome, multiplemyeloma, pancreatic- and cardiac-related amyloidosis, chronichemodialysis anthropathy, and Finnish and Iowa amyloidosis.

In addition, nicotinic receptors have been implicated as playing a rolein the body's response to alcohol ingestion. Thus, agonists forα-7nAChR's can be used in the treatment of alcohol withdrawal and inanti-intoxication therapy. Thus, in accordance with an embodiment of theinvention, the methods are related to treating a patient for alcoholwithdrawal or treating a patient with anti-intoxication therapy.

Agonists for the α-7nAChR subtypes can also be used for neuroprotectionagainst damage associated with strokes and ischemia andglutamate-induced excitotoxicity. Thus, in accordance with an embodimentof the invention there is provided a method related to providing forneuroprotection against damage associated with strokes and ischemia andglutamate-induced excitotoxicity.

As noted above, agonists for the α-7nAChR subtypes can also be used inthe treatment of nicotine addiction, inducing smoking cessation,treating pain, and treating jetlag. Thus, in accordance with anembodiment of the invention there is provided a method related totreatment of suffering from nicotine addiction, pain, and/or jetlag, ora method of inducing smoking cessation.

The condition of memory impairment is manifested by impairment of theability to learn new information and/or the inability to recallpreviously learned information. Memory impairment is a primary symptomof dementia and can also be a symptom associated with such diseases asAlzheimer's disease, schizophrenia, Parkinson's disease, Huntington'sdisease, Pick's disease, Creutzfeld-Jakob disease, HIV, cardiovasculardisease, and head trauma as well as age-related cognitive decline.

Thus, in accordance with an embodiment of the invention there isprovided a method related to treating a patient suffering from, forexample, mild cognitive impairment (MCI), vascular dementia (VaD),age-associated cognitive decline (AACD), amnesia associatedw/open-heart-surgery, cardiac arrest, and/or general anesthesia, sleepdeprivation induced cognitive impairment, chronic fatigue syndrome,narcolepsy, AIDS-related dementia, epilepsy-related cognitiveimpairment, Down's syndrome, Alcoholism related dementia, drug/substanceinduced memory impairments, Dementia Puglistica (Boxer Syndrome),andanimal dementia (e.g. dogs, cats, horses, etc. ).

Screening Assays for Modulatory Agents and Assays for the Determinationof Nicotinic Alpha-7 Receptor Levels and/or Activities

This invention provides methods of screening agents, in vitro or in vivo(e.g., in cell-based assays or in animal models), to identify thoseagents that modulate (e.g., enhance, stimulate, restore, inhibit, block,stabilize, destabilize, increase, facilitate, up-regulate, activate,amplify, augment, induce, decrease, down-regulate, diminish, lessen,reduce, etc.) synthesis and/or activity of the nicotinic alpha-7receptors of the invention to a putative agent, in the presence orabsence of an nicotinic alpha-7 stimulatory agent, and measuring theactivity of the alpha-7, e.g., as indicated by Ca⁺⁺ influx, compared tothe activity in the absence of the putative agent,measuring the totalamount of charge flowing across the membrane of said cell, or measuringthe change in a calcium-sensitive dye present in said cell, in responseto said agent.

As used herein, a compound that “modulates the activity of a neuronalnicotinic AchR” refers to a compound that alters the activity ofnicotinic alpha-7 receptor so that activity of the nicotinic alpha-7receptor is different in the presence of the compound than in theabsence of the compound. In particular, such compounds include agonistsor antagonists. The term agonist refers to a substance, such asacetylcholine, that activates receptor function; and the term antagonistrefers to a substance that interferes with receptor function. Typically,the effect of an antagonist is observed as a blocking of activation byan agonist. Antagonists include competitive and non-competitiveantagonists. A competitive antagonist (or competitive blocker) interactswith or near the site specific for the agonist (e.g., ligand orneurotransmitter) for the same or closely situated site. Anon-competitive antagonist or blocker inactivates the functioning ofreceptor by interacting with a site other than the site that interactswith the agonist.

A “nicotinic cholinergic agonist” is a compound that binds to andactivates a nicotinic acetylcholine receptor. By “activates” is intendedthe elicitation of one or more pharmacological, physiological, orelectrophysiological response. Such a response includes, but is notlimited to, cell membrane depolarization and increased permeability toCa⁺⁺ and other cations. Agents can also be partial agonists, e.g., whenan agent is only partly effective as an agonist.

A “nicotinic cholinergic antagonist” is a substance that binds to anicotinic acetylcholine receptor and prevents agonists from activatingthe receptor. Pure antagonists do not activate the receptor, but somesubstances may have mixed agonist and antagonist properties. Nicotiniccholinergic channel blockers block the ability of agonists to elicitcurrent flow through the nicotinic acetylcholine receptor channel, butdo so by blocking the channel rather than by preventing agonists frombinding to and activating the receptor.

A “nicotinic cholinergic receptor” intends a substance that influencesthe activity of the nicotinic acetylcholine receptor through interactionat one or more sites other than the classic agonist binding site. Theregulator may itself increase or decrease receptor activity, or mayinfluence agonist activity (for example, potentiating responses) withoutitself eliciting an overt change in channel current. A single substancecan have different properties at different nicotinic acetylcholinereceptor subtypes, for example, being an agonist at one receptor andantagonist at another, or an antagonist at one and a channel blocker atanother.

By “nAChr modulator” is intended a substance that may act as an agonist,antagonist, channel blocker, or regulator.

As understood by those of skill in the art, assay methods foridentifying compounds that modulate rhesus monkey neuronal nicotinicalpha-7 activity (e.g., agonists and antagonists) generally requirecomparison to a control. One type of “control” cell or “control” cultureis a cell or culture that is treated substantially the same as the cellor culture exposed to the test compound, except the control culture isnot exposed to test compound.

In another embodiment, this invention provides methods of screeningagents, in vitro or in vivo (e.g., in cell-based assays or in animalmodels), to identify those agents that modulate (e.g., enhance,stimulate, restore, inhibit, block, stabilize, destabilize, increase,facilitate, up-regulate, activate, amplify, augment, induce, decrease,down-regulate, diminish, lessen, reduce, etc.) expression of thenicotinic alpha-7 receptors of the invention.

In a further embodiment, this invention provides methods of screeningagents, in vitro or in vivo (e.g., in cell-based assays or in animalmodels), to identify those agents that modulate (e.g., enhance,stimulate, restore, inhibit, block, stabilize, destabilize, increase,facilitate, up-regulate, activate, amplify, augment, induce, decrease,down-regulate, diminish, lessen, reduce, etc.) the transport of analpha-7 subunit, or of a receptor comprising at least one such alpha-7subunit, to the cell membrane.

Assays for Ca⁺⁺ uptake can be performed either in the absence of aligand or following stimulation by an appropriate ligand. Appropriatestimulatory ligands include, e.g., nicotine or nicotinic acid(preferably the (−) enantiomer), carbamyl choline, cytosine,acetylcholine, epibatidine, or alpha-7 specific ligands such as GTS-21,4OH-GTS-21. Non alpha-7 specific ligands stimulation can be shown by thereduction or blocking of the signal with conventional inhibitors suchas, e.g., methyllycaconitine or alpha-bungarotoxin. Calcium uptake canbe measured conventionally, e.g., using calcium-sensitive dyes, such asFluo-3AM, Fluo-3, Fluo-4, Fluo-4AM, Rhod-2, Calcium Green-1. CalciumGreen-2, etc. See, also the examples below.

Among the types of modulatory agents that can be tested and identifiedby the methods of the invention are, e.g., small chemical compounds(e.g., inorganic or organic molecules, such as conventionalcombinatorial libraries), polypeptides, peptides, or peptide analogs,polynucleotides, antibodies that bind specifically to the polypeptidesof the invention, or the like.

Without wishing to be bound to any particular mechanism, it is proposedthat an inhibitory or stimulatory agent may act on the ligand bindingmoiety of the alpha-7 receptor, on an allosteric binding moiety, or onan element of the ion channel, thereby modulating activity of theprotein; or the agent may enter cells and, e.g., bind directly to theDNA neighboring the sequences coding for the polypeptides of theinvention, thereby increasing or decreasing their expression; or theagent may enter the cell and affect post-transcriptional processing,thereby modulating the protein activity; or the agent may affect thetransport of the alpha-7 to the cell membrane.

Any of the assays described herein can, of course, be adapted to any ofa variety of high throughput methodologies, as can the generation,identification and characterization of putative inhibitory orstimulatory agents. Agents identified on the basis of their ability tomodulate alpha-7 receptor expression or activity may also be used formodulating other neuronal nicotinic acetylcholine receptors, and/or fordiagnosing or treating disease conditions related thereto.

Antisense Oligonucleotides and Ribozymes

Potential antagonists or inhibitors of the invention include isolatedantisense oligonucleotides, or antisense constructs which expressantisense oligonucleotides, both of which classes of molecules can beprepared using conventional technology. Antisense technology can be usedto control gene expression through methods based on binding of apolynucleotide to DNA or RNA. Without wishing to be bound to anyparticular mechanism, types of antisense oligonucleotides and proposedmechanisms by which they function include, e.g., the following: The 5′coding portion of a polynucleotide sequence which encodes for a maturepolypeptide of the present invention can be used to design an antisenseoligonucleotide (e.g., an RNA, DNA, PNA etc. oligonucleotide) of anysite which is compatible with the objects of the invention, e.g., offrom about 10 to 40 base pairs in length. The antisense oligonucleotidecan hybridize to the mRNA and block translation of the mRNA moleculeinto an alpha-7 receptor polypeptide (see e.g., Okano, J. Neurochem.,56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1988)). Alternatively, anoligonucleotide can be designed to be complementary to a region of thegene involved in transcription (see, e.g, Lee et al., Nucl. Acids Res.,6:3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervan et al.,Science, 251: 1360 (1991)), thereby preventing transcription and theproduction of alpha-7 receptors. For further guidance on administeringand designing antisense, see, e.g., U.S. Pat. Nos. 6,200,960, 6,200,807,6,197,584, 6,190,869, 6,190,661, 6,187,587, 6,168,950, 6,153,595,6,150,162, 6,133,246, 6,117,847, 6,096,722, 6,087,343, 6,040,296,6,005,095, 5,998,383, 5,994,230, 5,891,725, 5,885,970, and 5,840,708.

Antisense polynucleotides can comprise modified, nonnaturally-occurringnucleotides and linkages between the nucleotides (e.g., modification ofthe phosphate-sugar backbone; methyl phosphonate, phosphorothioate, orphosphorodithioate linkages; and 2′-O-methyl ribose sugar units), e.g.,to enhance in vivo or in vitro stability, to confer nuclease resistance,to modulate uptake, to modulate cellular distribution andcompartmentalization, etc. Any effective nucleotide or modification canbe used, including those already mentioned, as known in the art, etc.,e.g., disclosed in U.S. Pat. Nos. 6,133,438; 6,127,533; 6,124,445;6,121,437; 5,218,103 (e.g., nucleoside thiophosphoramidites); 4,973,679;Sproat et al., “2′-O-Methyloligoribonucleotides: synthesis andapplications,” Oligonucleotides and Analogs A Practical Approach,Eckstein (ed.), IRL Press, Oxford, 1991, 49-86; Iribarren et al.,“2′-O-Alkyl Oligoribonucleotides as Antisense Probes,” Proc. Natl. Acad.Sci. USA, 1990, 87, 7747-7751; Cotton et al., “2′-O-methyl, 2′-O-ethyloligoribonucleotides and phosphorothioate oligodeoxyribonucleotides asinhibitors of the in vitro U7 snRNP-dependent mRNA processing event,”Nucl. Acids Res., 1991, 19, 2629-2635. Effective amounts of antisenseoligonucleotides as described above can be administered to a patient inneed thereof by conventional means.

Antisense oligonucleotides can also be delivered to cells via, e.g.,plasmids or other vectors, wherein the antisense sequence is operablylinked to an expression control sequence. In this manner, RNA or DNAantisense is expressed in a cell and inhibits production of alpha-7receptors. A total length of about 36 nucleotides can be used in cellculture with cationic lipisomes to facilitate cellular uptake, but forin vivo use, preferably shorter oligonucleotides are administered, e.g.,about 25 nucleotides.

In another embodiment, ribozymes corresponding to specific sequences canbe introduced into cells such that they cleave nicotinic alpha-7receptor coding or regulatory sequences. Ribozymes are enzymatic RNAmolecules capable of catalyzing the specific cleavage of RNA. Themechanism of ribozyme action involves sequence specific hybridization ofthe ribozyme molecule to complementary target RNA, followed by anendonucleolytic cleavage. Ribozyme molecules designed to catalyticallycleave target gene mRNA transcripts can also be used to preventtranslation of target gene mRNA and expression of target gene. (See,e.g., PCT International Publication WO90/11364, published Oct. 4, 1990;Sarver et al., 1990, Science 247:1222-1225). While ribozymes that cleavemRNA at site specific recognition sequences can be used to destroytarget gene mRNAs, the use of hammerhead ribozymes is preferred.Hammerhead ribozymes cleave mRNAs at locations dictated by flankingregions that form complementary base pairs with the target mRNA. Thesole requirement is that the target mRNA have the following sequence oftwo bases: 5′-UG-3′. The construction and production of hammerheadribozymes is well known in the art and is described more fully inHaseloff and Gerlach, 1988, Nature, 334:585-591. Preferably the ribozymeis engineered so that the cleavage recognition site is located near the5′ end of the target mRNA, i.e., to increase efficiency and minimize theintracellular accumulation of non-functional mRNA transcripts.

The ribozymes of the present invention also include RNAendoribonucleases (hereinafter “Cech-type ribozymes”) such as the onewhich occurs naturally in Tetrahymena Thermophila (known as the IVS, orL-19 IVS RNA) and which has been extensively described by Thomas Cechand collaborators (Zaug, et al., 1984, Science, 224:574-578; Zaug andCech, 1986, Science, 231:470-475; Zaug, et al., 1986, Nature,324:429-433; published International patent application No. WO 88/04300by University Patents Inc.; Been and Cech, 1986, Cell, 47:207-216). TheCech-type ribozymes have an eight base pair active site which hybridizesto a target RNA sequence whereafter cleavage of the target RNA takesplace. The invention encompasses those Cech-type ribozymes which targeteight base-pair active site sequences that are present in target gene.

As in the antisense approach, the ribozymes can be composed of modifiedoligonucleotides (e.g., for improved stability, targeting, etc.) andshould be delivered to cells which express the target gene in vivo. Apreferred method of delivery involves using a DNA construct “encoding”the ribozyme under the control of a strong constitutive pol III or polII promoter, so that transfected cells will produce sufficientquantities of the ribozyme to destroy endogenous target gene messagesand inhibit translation. Because ribozymes, unlike antisense molecules,are catalytic, a lower intracellular concentration is required forefficiency.

Diagnostics/Assays for Alpha-7 Receptor Polypeptides

The present invention provides for a means of diagnosing or stagingactual or potential disease conditions involving altered levels ofnicotinic alpha-7 receptors by determining the amounts (e.g., thepresence or absence, or the quantity) of the polypeptides of theinvention, or their levels of activity, in an animal suspected of havingsuch a disease condition or being at risk therefor. For example, theinvention provides a process for diagnosing a disease in an animalafflicted therewith, or diagnosing a susceptibility to a disease in ananimal at risk thereof, wherein said disease is related, for example, toan over- or under-expression or activity of av alpha-7 receptoraccording to the invention, comprising determining the amount of saidalpha-7 receptor or the level of said alpha-7 receptor activity in acell from said animal, wherein said animal is preferably a mammal, mostpreferably a primate.

When assaying samples for diagnostic purposes, using any of the methodsdescribed herein, samples may be obtained from any suitable cell,tissue, organ, or bodily fluid from a patient, including but not limitedto blood, urine, saliva, tissue biopsy and autopsy material. In oneembodiment, samples for diagnosis are taken from cells or tissues inwhich high levels of alpha-7 receptor expression are normally observed,e.g., neurological tissue. In a preferred embodiment, the diseaseconditions to be diagnosed involve loss of memory as a primary orsecondary effect thereof, especially loss of long term memory, and thecells tested are typically neurons, especially those of the brain, forexample, neurons of the hippocampal region (e.g., in hippocampalslices).

Enzymatic assays for the various activities exhibited by nicotinicalpha-7 receptors are conventional. Some such assays are describedabove. Detection and/or quantitation of protein levels can beaccomplished by any of a variety of conventional methods, e.g., methodsbased on antibodies or antigen-specific fragments of the invention.Immunological assays include, e.g., ELISA, RIA and FACS assays. Atwo-site, monoclonal-based immunoassay, utilizing antibodies reactive totwo non-interfering epitopes on a alpha-7 receptor polypeptide arepreferred, but a competitive binding assay may be employed. These andother assays are described, e.g., in Hampton et al. (1990). SerologicalMethods, a Laboratory Manual, APS Press, St. Paul, Minn.

The invention provides methods for diagnosing a disease orsusceptibility thereto wherein said disease is related to production ofan aberrant form of an alpha-7 receptor according to the invention,e.g., one resulting from a genetic mutation. Such aberrant (or variant)proteins include those described above, e.g., proteins having amino acidsubstitutions, deletions, inversions, insertions, rearrangements (e.g.,as a result of aberrant splicing events) or inappropriatepost-translational modifications. Aberrant proteins may exhibitincreased or decreased activity of any of the functions describedelsewhere herein. Aberrant proteins may also exhibit increased ordecreased interactions with other proteins, such as, e.g.,proteinkinases, cytoskeletal proteins, etc.

Variant proteins (e.g., mutants or muteins) can be detected by any of avariety of conventional methods. For example, antibodies or antigenbinding fragments can be used to detect the presence of aberrant formsof the polypeptides disclosed herein, using immunological methods suchas those described above.

In accordance with the present invention, an antibody or antigen-bindingfragment can be present in a kit, where the kit includes, e.g., one ormore antibodies or antigen-binding fragments, a desired buffer,detection compositions, proteins (e.g., wild type) to be used ascontrols, etc.

Diagnostic/Assays for Alpha-7 Receptor Nucleic Acid

Assays involving polynucleotides can be used to determine the presenceor absence of a nucleic acid in a sample and/or to quantify it, or todetect a mutation or polymorphism. Such assays can be used, e.g., fordiagnostic, prognostic, research, or forensic purposes. The assays canbe, e.g., membrane-based, solution-based, or chip-based. Assays can beperformed at the single-cell level, or in a sample comprising manycells, where the assay is “averaging” expression over the entirecollection of cells and tissue present in the sample.

Any suitable assay format can be used, including, but not limited to,Southern blot analysis, Northern blot analysis, polymerase chainreaction (“PCR”) (e.g., Saiki et al., Science, 241:53, 1988; U.S. Pat.Nos. 4,683,195, 4,683,202, and 6,040,166; PCR Protocols: A Guide toMethods and Applications, Innis et al., eds., Academic Press, New York,1990), reverse transcriptase polymerase chain reaction (“RT-PCR”),anchored PCR, rapid amplification of cDNA ends (“RACE”) (e.g., Schaeferin Gene Cloning and Analysis: Current Innovations, Pages 99-115, 1997),ligase chain reaction (“LCR”) (EP 320 308), one-sided PCR (Ohara et al.,Proc. Natl. Acad. Sci., 86:5673-5677, 1989), indexing methods (e.g.,U.S. Pat. No. 5,508,169), in situ hybridization, differential display(e.g., Liang et al., Nucl. Acid. Res., 21:3269-3275, 1993; U.S. Pat.Nos. 5,262,311, 5,599,672 and 5,965,409; WO97/18454; Prashar andWeissman, Proc. Natl. Acad. Sci., 93:659-663, and U.S. Pat. Nos.6,010,850 and 5,712,126; Welsh et al., Nucleic Acid Res., 20:4965-4970,1992, and U.S. Pat. No. 5,487,985) and other RNA fingerprintingtechniques, nucleic acid sequence based amplification (“NASBA”) andother transcription based amplification systems (e.g., U.S. Pat. Nos.5,409,818 and 5,554,527; WO 88/10315), polynucleotide arrays (e.g., U.S.Pat. Nos. 5,143,854, 5,424,186; 5,700,637, 5,874,219, and 6,054,270; PCTWO 92/10092; PCT WO 90/15070), QBeta Replicase (PCT/US87/00880), StrandDisplacement Amplification (“SDA”), Repair Chain Reaction (“RCR”),nuclease protection assays, subtraction-based methods, Rapid-Scan™, etc.Additional useful methods include, but are not limited to, e.g.,template-based amplification methods, competitive PCR (e.g., U.S. Pat.No. 5,747,251), redox-based assays (e.g., U.S. Pat. No. 5,871,918),Taqman-based assays (e.g., Holland et al., Proc. Natl. Acad, Sci.,88:7276-7280, 1991; U.S. Pat. Nos. 5,210,015 and 5,994,063), real-timefluorescence-based monitoring (e.g., U.S. Pat. 5,928,907), molecularenergy transfer labels (e.g., U.S. Pat. Nos. 5,348,853, 5,532,129,5,565,322, 6,030,787, and 6,117,635; Tyagi and Kramer, Nature Biotech.,14:303-309, 1996). Any method suitable for single cell analysis of geneor protein expression can be used, including in situ hybridization,immunocytochemistry, MACS, FACS, flow cytometry, etc. For single cellassays, expression products can be measured using antibodies, PCR, orother types of nucleic acid amplification (e.g, Brady et al., MethodsMol. & Cell. Biol. 2, 17-25, 1990; Eberwine et al., 1992, Proc. Natl.Acad. Sci., 89, 3010-3014, 1992; U.S. Pat. No. 5,723,290). These andother methods can be carried out conventionally, e.g., as described inthe mentioned publications.

The invention provides methods for diagnosing a disease in an animalafflicted therewith, or diagnosing susceptibility to a disease in ananimal at risk thereof, wherein said disease is related, for example, toan over- or under-expression of a polynucleotide encoding an alpha-7receptor according to the invention, comprising determining the amountof said polynucleotide in a cell from said animal, wherein said animalis preferably a mammal and most preferably a primate. Any of the assaymethods described herein, or otherwise known in the art, can be used todetermine the presence of and/or to quantitate, such polynucleotides.

Furthermore, detection of a mutated or polymorphic form of a gene allowsa diagnosis of a disease or a susceptibility to a disease, which resultsfrom expression of a mutated alpha-7 receptor polypeptide that may have,for example, increased or decreased activity in certain tissues. Suchmutations include, e.g., any of those described elsewhere herein, e.g.,point mutations, insertions, deletions, substitutions, transversions,and chromosomal translocations.

Individuals carrying mutations in a gene of the present invention may bedetected at the DNA level by a variety of techniques. Genomic DNA may beused directly for detection or may be amplified enzymatically by usingPCR (Saiki et al., Nature, 324:163-166 (1986); Innis et al eds., (1996)PCR Protocols: A Guide to Methods in Amplification, Academic Press, NewYork) prior to analysis. RNA or cDNA may also be used for the samepurpose. As an example, PCR primers complementary to the nucleic acidencoding an alpha-7 receptor can be used to identify and analyzemutations. For example, deletions and insertions can be detected by achange in size of the amplified product in comparison to the normalgenotype. Point mutations can be identified, e.g., by hybridizingamplified DNA to radiolabeled RNA or radiolabeled antisense DNAsequences. Perfectly matched sequences can be distinguished frommismatched duplexes by a variety of methods, including, e.g., RNase Adigestion or by differences in melting temperatures. Rapid sequencingmethods can be employed.

Sequence differences between the reference gene and genes havingmutations may be revealed by the direct DNA sequencing method. Inaddition, cloned DNA segments may be employed as probes to detectspecific DNA segments. The sensitivity of this method is greatlyenhanced when combined with PCR. For example, a sequencing primer isused with double-stranded PCR product or a single-stranded templatemolecule generated by a modified PCR. The sequence determination isperformed by conventional procedures with radiolabeled nucleotide or byautomatic sequencing procedures with fluorescent-tags.

A polynucleotide sequence coding for part or all of an alpha-7 receptorof the invention may act as a reference for the development of probes,e.g., as long as 30 to 45 nucleotides, or longer, that can be used toprobe the genome of animals suspected of being at risk for disease, orhaving such disease. Probes corresponding to regulatory sequences e.g.,sequences which govern the amount of mRNA coding for the alpha-7receptor of the invention, or of the alpha-7 receptor protein produced,can also be used. Such regulatory sequences include, e.g., promoter orenhancer elements, sequences which govern splicing events, stability ofnucleic acid or protein, termination/polyadenylation and/orintracellular localization of mRNAs or proteins.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230:1242 (1985)), or by mass spectroscopy analysis.

In addition, sequence changes at specific locations may also be revealedby nuclease protection assays, such as RNase and S1 protection or thechemical cleavage method (e.g, Cotton et al., PNAS, USA, 85:4397-4401(1985)) and these are deemed within the methods of the invention.

Thus, the detection of a specific DNA sequence may be achieved bymethods such as, e.g, hybridization, RNase protection, chemicalcleavage, direct DNA sequencing or the use of restriction enzymes,(e.g., Restriction Fragment Length Polymorphisms (RFLP)) and Southernblotting of genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations can also be detected by in situ analysis.

Mutations in regulatory elements can also affect the level ofpolynucleotide (e.g., mRNA) or protein made, and can give rise todisease symptoms. Such mutations include, e.g., mutations in promoter orenhancer elements, splice signals, termination and/or polyadenylationsignals; mutations which result in truncated proteins, such as chainterminators; sites involved in feed-back regulation of nucleic acid orpolypeptide production; etc. Diagnostic methods to detect such mutationsin regulatory elements are conventional.

In accordance with the present invention, a polynucleotide can bepresent in a kit, where the kit includes, e.g., one or morepolynucleotides, a desired buffer (e.g., phosphate, tris, etc.),detection compositions, RNA or cDNA from different tissues to be used ascontrols, libraries, etc. The polynucleotide can be labeled orunlabeled, with radioactive or non-radioactive labels as known in theart. Kits can comprise one or more pairs of polynucleotides foramplifying nucleic acids specific for an alpha-7 receptor, e.g.,comprising a forward and reverse primer effective in PCR. These includeboth sense and anti-sense orientations. For instance, in PCR-basedmethods (such as RT-PCR), a pair of primers are typically used, onehaving a sense sequence and the other having an antisense sequence.

Other Uses of Polynucleotides

The sequences of the present invention are also valuable for chromosomeidentification. The polynucleotides coding for the alpha-7 receptor ofthe invention, and homologs thereof, are specifically targeted to andcan hybridize with a particular location on an individual chromosome.Moreover, there is a current need for identifying particular sites onthe chromosome, for example, as part of a genome project. Thus,sequences can be mapped to chromosomes, e.g., by preparing PCR primers(preferably 15-25 bp) from the cDNA.

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphasechromosomal spread can likewise be used to provide a precise chromosomallocation in one step. This technique can be used with cDNA having atleast 50 or 60 bases. For a review of this technique, see Verma et al.,Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, NewYork (1988). The chromosomal location of the alpha-7 receptor genes isknown to those skilled in the art.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man (available on line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

One can determine the differences in the cDNA or genomic sequencebetween affected and unaffected individuals. If a mutation is observedin some or all of the affected individuals but not in any normalindividuals, then the mutation is likely to be a causative agent of thedisease. With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

A fragment of a polynucleotide of the present invention may also be usedas a hybridization probe, e.g, for a cDNA or genomic library to isolatea full length cDNA (or genomic DNA) and to isolate other cDNAs (orgenomic DNAs) which have a high sequence similarity to the gene orsimilar biological activity. Probes of this type preferably have atleast 7 or 8 bases, more preferably about 10, 11, 12, 13, 14 15, 20, 25,etc bases, and most preferably at least about 30 bases, and exhibitabout 65-100% sequence identity to part or all of the sequence codingfor the novel alpha-7 receptor disclosed in FIG. 1, 3, or 5 (SEQ ID NOS:1, 3, or 5). Such probes may also have 45 or more bases but againcontain sequences which exhibit about 65-100% sequence identity to asequence coding for some or all of the nicotinic alpha-7 receptorpolypeptide of the invention, or a variant thereof. Because of thedegeneracy of the genetic code, many sequences exist which exhibit ahigh degree of sequence identity to sequences coding for part or all ofa novel alpha-7 receptor disclosed herein. The set of such sequencesalso includes those that code for amino acid sequences that arethemselves homologous to part or all of the nicotinic alpha-7 receptor.Hybridization probes are specific to, or for, a selected polynucleotide.The phrases “specific for” or “specific to” a polynucleotide have afunctional meaning that the probe can be used to identify the presenceof one or more target genes in a sample. The probe is specific in thesense that it can be used to detect a polynucleotide above backgroundnoise (“non-specific binding”).

Therapeutics

The methods of the present invention are also directed to facilitatingthe development of potentially useful therapeutic agents that may beeffective in combating alpha-7 receptor mediated or related diseases orconditions, and to methods of effecting such treatments. The inventionalso provides methods to enhance or restore memory function in “normal”subjects, e.g., by activating brain, especially hippocampal, neuronalalpha-7 receptors.

Any agent which modulates the expression and/or activity of an alpha-7receptor polypeptide or polynucleotide of the invention can be usedtherapeutically. Some such agents are discussed elsewhere herein.

Agents which affect expression and/or activities of polypeptides of theinvention can be administered to patients in need thereof byconventional procedures, in order to prevent or treat disease conditionsas disclosed elsewhere herein and/or to ameliorate symptoms of thoseconditions. Such agents can be formulated into pharmaceuticalcompositions comprising pharmaceutically acceptable excipients,carriers, etc., using conventional methodologies. Formulations andexcipients which enhance transfer (promote penetration) of an agentacross the blood-brain barrier are also well-known in the art.

In addition to agents which can moderate the expression or activity ofan alpha-7 receptor, treatment methods according to the invention alsoencompass the administration of an alpha-7 receptor or variant orfragment thereof to a patient in need of such therapy. For example, sucha polypeptide or fragment can compensate for reduced or aberrantexpression or activity of the protein, and/or, by virtue of, e.g.,higher affinity for a target, can provide effective competition for it.In another embodiment, conventional methods of immunotherapy can beused.

Polynucleotides of the invention can also be used in methods of genetherapy, e.g., utilized in gene delivery vehicles. The gene deliveryvehicle may be of viral or non-viral origin (see generally, Jolly,Cancer Gene Therapy 1:51-64 (1994) Kimura, Human Gene Therapy 5:845-852(1994); Connelly, Human Gene Therapy 1:185-193 (1995); and Kaplitt,Nature Genetics 6:148-153 (1994). Gene therapy vehicles for delivery ofconstructs including a coding sequence of a therapeutic of the inventioncan be administered either locally or systemically. These constructs canutilize viral or non-viral vector approaches. Expression of such codingsequences can be induced using endogenous mammalian or heterologouspromoters. Expression of the coding sequence can be either constitutiveor regulated.

The present invention can employ recombinant retroviruses which areconstructed to carry or express a selected nucleic acid molecule ofinterest. Retrovirus vectors that can be employed include thosedescribed in EP 0 415 731; WO 90/07936; WO 94/03622; WO 93/25698; WO93/25234; U.S. Pat. No. 5,219,740; WO 93/11230; WO 93/10218; Vile andHart, Cancer Res. 53:3860-3864 (1993); Vile and Hart, Cancer Res.53:962-967 (1993); Ram et al., Cancer Res. 53:83-88 (1993); Takamiya etal., J. Neurosci. Res. 33:493-503 (1992); Baba et al., J. Neurosurg.79:729-735 (1993); U.S. Pat. No. 4,777,127; GB Patent No. 2,200,651; andEP 0 345 242. Preferred recombinant retroviruses include those describedin WO 91/02805.

Packaging cell lines suitable for use with the above-describedretroviral vector constructs may be readily prepared (see PCTpublications WO 95/30763 and WO 92/05266), and used to create producercell lines (also termed vector cell lines) for the production ofrecombinant vector particles. Within particularly preferred embodimentsof the invention, packaging cell lines are made from human (such asHT1080 cells) or mink parent cell lines, thereby allowing production ofrecombinant retroviruses that can survive inactivation in human serum.

The present invention also employs aphavirus-based vectors that canfunction as gene delivery vehicles. Such vectors can be constructed froma wide variety of alphaviruses, including, for example, Sindbis virusvectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross Rivervirus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitisvirus (ATCC VR-923; ATCC VR-1250 ATCC VR-1249; ATCC VR-532).Representative examples of such vector systems include those describedin U.S. Pat. Nos. 5,091,309; 5,217,879; and 5,185,440; and PCTPublication Nos. WO 92/10578; WO 94/21792; WO 95/27069; WO 95/27044; andWO 95/07994.

Gene delivery vehicles of the present invention can also employparvovirus such as adeno-associated virus (AAV) vectors. Representativeexamples include the AAV vectors disclosed by Srivastava in WO 93/09239,Samulski et al., J. Vir. 63:3822-3828 (1989); endelson et al., Virol.166:154-165 (1988); and Flotte et al., P.N.A.S. 90:10613-10617 (1993).

Representative examples of adenoviral vectors include those described byBerkner, Biotechniques 6:616-627 (Biotechniques); Rosenfeld et al.,Science 252:431-434 (1991); WO 93/19191; Kolls et al., P.N.A.S. 215-219(1994); Kass-Eisler et al., P.N.A.S. 90:11498-11502 (1993); Guzman etal., Circulation 88:2838-2848 (1993); Guzman et al., Cir. Res.73:1202-1207 (1993); Zabner et al., Cell 75:207-216 (1993); Li et al.,Hum. Gene Ther. 4:403-409 (1993); Cailaud et al., Eur. J. Neurosci. 5:1287-1291 (1993); Vincent et al., Nat. Genet. 5:130-134 (1993); Jaffe etal., Nat. Genet. 1:372-378 (1992); and Levrero et al., Gene 101:195-202(1992). Exemplary adenoviral gene therapy vectors employable in thisinvention also include those described in WO 94/12649, WO 93/03769; WO93/19191; WO 94/28938; WO 95/11984 and WO 95/00655. Administration ofDNA linked to killed adenovirus as described in Curiel, Hum. Gene Ther.3:147-154 (1992), may be employed.

Other gene delivery vehicles and methods may be employed, includingpolycationic condensed DNA linked or unlinked to killed adenovirusalone, for example, Curiel, Hum. Gene Ther. 3:147-154 (1992);ligand-linked DNA, for example, see Wu, J. Biol. Chem. 264:16985-16987(1989); eukaryotic cell delivery vehicles cells, for example see U.S.Ser. No. 08/240,030, filed May 9, 1994, and U.S. Ser. No. 08/404,796;deposition of photopolymerized hydrogel materials; hand-held genetransfer particle gun, as described in U.S. Pat. No. 5,149,655; ionizingradiation as described in U.S. Pat. No. 5,206,152 and in WO 92/11033;nucleic charge neutralization or fusion with cell membranes. Additionalapproaches are described in Philip, Mol. Cell Biol. 14:2411-2418 (1994)and in Woffendin, Proc. Natl. Acad. Sci. 91:1581-1585 (1994).

Naked DNA may also be employed. Exemplary naked DNA introduction methodsare described in WO 90/11092 and U.S. Pat. No. 5,580,859. Uptakeefficiency may be improved using biodegradable latex beads. DNA coatedlatex beads are efficiently transported into cells after endocytosisinitiation by beads. The method may be improved further by treatment ofthe beads to increase hydrophobicity and thereby facilitate disruptionof the endosome and release of the DNA into thr cytoplasm. Liposomesthat can act as gene delivery vehicles are described in U.S. Pat. No.5,422,120, PCT Patent Publication Nos. WO 95/13796, WO 94/23697 and WO91/14445, and EP No. 0 524 968.

Further non-viral delivery suitable for use includes mechanical deliverysystems such as the approach described in Woffendin et al., Proc. Natl.Acad. Sci. USA 91(24):11581-11585 (1994). Moreover, the coding sequenceand the product of expression of such can be delivered throughdeposition of photopolymerized hydrogel materials. Other conventionalmethods for gene delivery that can be used for delivery of the codingsequence include, for example, use of hand-held gene transfer particlegun, as described in U.S. Pat. No. 5,149,655; use of ionizing radiationfor activating transferred gene, as described in U.S. Pat. No. 5,206,152and PCT Patent Publication No. WO 92/11033.

Computer-based Applications

The nucleotide or amino acid sequences of the invention are alsoprovided in a variety of media to facilitate use thereof. As usedherein, “provided” refers to a manufacture, other than an isolatednucleic acid or amino acid molecule, which contains a nucleotide oramino acid sequence of the present invention. Such a manufactureprovides the nucleotide or amino acid sequences, or a subset thereof(e.g., a subset of open reading frames (ORFs)) in a form which allows askilled artisan to examine the manufacture using means not directlyapplicable to examining the nucleotide or amino acid sequences, or asubset thereof, as they exist in nature or in purified form.

In one application of this embodiment, a nucleotide or amino acidsequence of the present invention can be recorded on computer readablemedia. As used herein, “computer readable media” refers to any mediumthat can be read and accessed directly by a computer. Such mediainclude, but are not limited to: magnetic storage media, such as floppydiscs, hard disc storage medium, and magnetic tape; optical storagemedia such as CD-ROM; electrical storage media such as RAM and ROM; andhybrids of these categories such as magnetic/optical storage media. Theskilled artisan will readily appreciate how any of the presently knowncomputer readable mediums can be used to create a manufacture comprisingcomputer readable medium having recorded thereon a nucleotide or aminoacid sequence of the present invention.

As used herein, “recorded” refers to a process for storing informationon computer readable medium. The skilled artisan can readily adopt anyof the presently known methods for recording information on computerreadable medium to generate manufactures comprising the nucleotide oramino acid sequence information of the present invention.

A variety of data storage structures are available to a skilled artisanfor creating a computer readable medium having recorded thereon anucleotide or amino acid sequence of the present invention. The choiceof the data storage structure will generally be based on the meanschosen to access the stored information. In addition, a variety of dataprocessor programs and formats can be used to store the nucleotidesequence information of the present invention on computer readablemedium. The sequence information can be represented in a word processingtext file, formatted in commercially-available software such asWordPerfect and Microsoft Word, or represented in the form of an ASCIIfile, stored in a database application, such as DB2, Sybase, Oracle, orthe like. The skilled artisan can readily adapt any number ofdataprocessor structuring formats (e.g., text file or database) in orderto obtain computer readable medium having recorded thereon thenucleotide sequence information of the present invention.

By providing the nucleotide or amino acid sequences of the invention incomputer readable form, the skilled artisan can routinely access thesequence information for a variety of purposes. For example, one skilledin the art can use the nucleotide or amino acid sequences of theinvention in computer readable form to compare a target sequence ortarget structural motif with the sequence information stored within thedata storage means. Search means are used to identify fragments orregions of the sequences of the invention which match a particulartarget sequence or target motif.

As used herein, a “target sequence” can be any DNA or amino acidsequence of six or more nucleotides or two or more amino acids. Askilled artisan can readily recognize that the longer a target sequenceis, the less likely a target sequence will be present as a randomoccurrence in the database. The most preferred sequence length of atarget sequence is from about 10 to 100 amino acids or from about 30 to300 nucleotide residues. However, it is well recognized thatcommercially important fragments, such as sequence fragments involved ingene expression and protein processing, may be of shorter length.

As used herein, “a target structural motif,” or “target motif,” refersto any rationally selected sequence or combination of sequences in whichthe sequence(s) are chosen on a three-dimensional configuration which isformed upon the folding of the target motif. There are a variety oftarget motifs known in the art. Protein target motifs include, but arenot limited to, enzyme active sites and signal sequences. Nucleic acidtarget motifs include, but are not limited to, promoter sequences,hairpin structures and inducible expression elements (protein bindingsequences).

Computer software is publicly available which allows a skilled artisanto access sequence information provided in a computer readable mediumfor analysis and comparison to other sequences. A variety of knownalgorithms are disclosed publicly and a variety of commerciallyavailable software for conducting search means are and can be used inthe computer-based systems of the present invention. Examples of suchsoftware includes, but is not limited to, MacPattern (EMBL), BLASTN andBLASTX (NCBIA).

For example, software which implements the BLAST (Altschul et al. (1990)J. Mol. Biol. 215:403-410) and BLAZE (Brutlag et al. (1993) Comp. Chem.17:203-207) search algorithms on a Sybase system can be used to identifyopen reading frames (ORFs) of the sequences of the invention whichcontain homology to ORFs or proteins from other libraries. Such ORFs areprotein encoding fragments and are useful in producing commerciallyimportant proteins such as enzymes used in various reactions and in theproduction of commercially useful metabolites.

In the foregoing and in the following examples, all temperatures are setforth uncorrected in degrees Celsius; and, unless otherwise indicated,all parts and percentages are by weight.

EXAMPLES Example 1 Determination of the 5′-, 3′-ends of Rhesus MonkeyNicotinic Alpha-7 Receptor Subunit

A Gene Racer cDNA library was generated using rhesus monkey brain mRNA(Biochain) according to the standard protocol (Invitrogen). Fourprimers, mkα7-5′R, mkα7-5′N, mkα7-3′R and mkα7-3′N, were designed basedon Genbank sequence AJ245976 and used to PCR the 5′ and 3′ endssequences of rhesus monkey alpha 7. RACE primers: (SEQ ID NO: 7)mkα7-5′R: 5′-CTCATCTCCACGCTGGCCAGGTGCAG (SEQ ID NO: 8) mkα7-3′R:5′-CATGAAGAGGCCGGGAGAGGATAAGGTGCG Nest primers: (SEQ ID NO: 9) mkα7-5′N:5′-CGCACCTTATCCTCTCCCGGCCTCTTCATG (SEQ ID NO: 10) mkα7-3′N:5′-CTGCACCTGGCCAGCGTGGAGATGAG

5′ and 3′ RACE PCR reactions were performed on rhesus monkey brain cDNAlibrary using mkα7-5′R, mkα7-3′R and Gene Racer 5′-, 3′-RACE primersrespectively. The PCR reactions were carried out using PCRx system andplatinum HF polymerase (Invitrogen) with the following cyclingcharacteristics: 94° C. for 3 minutes for 1 cycle; 94° C. for 30 secondsand 68° C. for 1 minute and 30 seconds for 35 cycles; 68 ° C for 7minutes for 1 cycle. After PCR, the resulting fragments were used astemplates for nested PCR using mkα7-5′N, mkα7-3′N and Gene Racer 5′-,3′-Nest primers respectively. The nested PCR protocol was the same asthe RACE PCRs.

The nested PCR products were then column purified (Qiagen) and clonedinto pcDNA3.1 v5/his TOPO vector and sequenced. One clone, namedmk-3′N#2, contains the 3′ end of rhesus monkey alpha 7 and is shownbelow. Another clone, named mkα7-5′N#13, contains the incomplete 5′ endof rhesus monkey alpha 7 and is shown below. mk-3′N#2: the 3′ stop codonand poly A+ signal are bolded. (SEQ ID NO: 11)CTGCAGCCTGGCCAGCGTGGAGATGAGCGCCGTGGCGCCGCCGCCTGCCAGCAACGGGAACCTGCTGTACATCGGCTTCCGCGGCCTGGACGGCATGCATTGCGCCCCGACCCCCGACTCCGGGGTGGTGTGCGGCCGCATGGCCTGCTCCCCCACGCACGACGAGCACCTCCTGCACGGTGGGCAGCCCCCCGAGGGGGACCCGGACCTGGCCAAGATCCTGGAGGAGGTCCGCTACATCGCCAACCGCTTTCGCTGCCAGGACGAAAGCGAGGCGGTCTGCAGCGAGTGGAAGTTCGCCGCCTGCGTGGTGGACCGCCTGTGCCTCATGGCCTTCTCGGTCTTCACCATCATCTGCACCATCGGCATCCTGATGTCGGCTCCCAACTTCGTGGAGGCCGTGTCCAAAGACTTTGCGTAACCACGCCTGGTTCTGTACATGTGGAAAACTCACAGATGGGCAAGGCCTCTGGCTTGGTGAGATTTTGGGGTGCTAATCCAGGACAACATTAAACGCCACAACTCCGATGTTCCCTTCTGGCTGTCAGTCGTGTCGCTCACGGTTTCCTCATTACTTTAGGTAGTAGGATCTCAGCACTCAGTTTAATACGCTCAGGTGGGCTGATGATCCCCTTGGCACATCCGCACTGTCGGTCAGCAGGGCCACTGAGAAGTCATTTTGCCCATTAGCCCACTGCCTGGAAAGCCCTTCAGAGAGCTCCCAGTGGCTCCTCACCCCGGGACAGTTGGTTTTGCATGTCTGCATGCCACTTGCCATGAAGGCCTACCTGAAAATTCAACATTTGCTTTTTGCTTGTGTACAAACCTAGATTGAAGCTAAAATAAACCAGACTCACTAAATCCAAAAAAAAAAAAAAAA mkα7-5′N#13: (SEQ ID NO: 12)GTATTTTGAGCGCGTCTCGATCAGCTTTCGTTTCAGTCTTCTGTTTCCGTCACCCACACGGGCATATTCAAGAGTTCCTGCTACATCGACGTGCGCCGGTTTCCCTTTGATGTGCAGCATTGCAAACTGAAGTTTGGATCCTGGTCTTATGGAGGCTGGTCCTTGGATCTGCAGATGCAGGAGGCAGATATCAGTGGCTATATCCCCAGTGGAGAATGGGACCTAGTGGGAATTCCCGGCAAGAGGAGTGAAAAGTTCTATGAGTGCTGCAAAGAGCCCTACCCCGATGTCACCTTCACAGTGACCATGCGCCGCAGGACCCTCTACTACGGCCTCAACCTGCTGATCCCCTGTGTGCTCATCTCTGCCCTTGCCCTGCTGGTGTTCCTGCTTCCTGCAGATTCCGGGGAGAAGATTTCCCTGGGGATAACAGTCTTACTCTCTCTCACTGTCTTCATGCTGCTCGTGGCTGAGATCATGCCCGCAACATCTGATTCAGTACCATTGATAGCCCAGTACTTCGCCAGCACCATGATCATCGTGGGCCTCTCCGTGGTGGTGACGGTGATCGTGCTGCAGTACCACCACCACGACCCCGACGGGGGCAAGATGCCCAAGTGGACCAGAGTCATCCTTCTGAACTGGTGCGCGTGGTTCCTGCGCATGAAGAGGCCGGGAGAGGATAAGGTGCG

To obtain the complete 5′ end sequence of rhesus monkey alpha 7, twoprimers, mkα7-5′R1 and mkα7-5′N1, were designed according to thesequence of mkα7-5′N#13 and used to PCR the same cDNA library asdescribed above, yet with the following cycling characteristics: 94° C.for 3 minutes for 1 cycle; 94° C. for 30 seconds, 65° C. for 30 secondsand 68° C. for 1 minute for 35 cycles; 68° C. for 7 minutes for 1 cycle.The nested PCR products were then column purified (Qiagen) and clonedinto pcDNA3.1 v5/his TOPO vector and sequenced. RACE primer (SEQ ID NO:13) mkα7-5′R1: 5′-GACCAGCCTCCATAAGACCAGGATCCAAACTTCAG Nest primer (SEQID NO: 14) mkα7-5′N1: 5′-CGCACGTCGATGTAGCAGGAACTCTTGAATATGC

One clone, named mkα7#1, still only contained the incomplete 5′ end ofrhesus monkey alpha-7 and is shown below. mkα7#1: (SEQ ID NO: 15)CATTGGCGGCATCTGTCCTCCCCGACAGGGTGCCTCCAGCACTTCAGATCCCAGCCGAGAGTCTGGCTGCTAGCGCCCAGCAAACGTGTCCCTGCAAGGCGAGTTCCAGAGGAAGCTTTACAAGGAGCTGGTCAAGAACTACAACCCCTTGGAGAGGCCCGTGGCCAATGACTCGCAACCGCTCACCGTCTACTTCTCCCTGAGCCTCCTGCAGATCATGGACGCGGATGAGAAGAACCAAGTTTTAACCACCAACATTTGGCTGCAAATGTCTTGGACAGATCACTATTTACAGTGGATGTGTCAGAATATCCAGGGGTGAAGACTGTTCGTTTCCCAGATGGCCAGATTTGGAAACCAGACATTCTTCTCTATAACAGTGCGGATGAGCGCTTTGACGCCACATTCCACACCAACGTGTTGGTGAATTCTTCTGGGCATTGCCAGTACCTGCCTCCAGGCATATTCAAGAGTTCCTGCTAATCGACGTGCG

Again, to obtain the complete 5′ end sequence of rhesus monkey alpha-7,two more primers, mkα7-5′R2 and mkα7-5′N2, were designed according tothe sequence of mkα7#1 and used to PCR the same cDNA library asdescribed above, yet with the following cycling characteristics: 94° C.for 5 minutes for 1 cycle; 94° C. for 20 seconds, 65° C. for 20 secondsand 68° C. for 30 seconds for 35 cycles; 68° C. for 7 minutes for 1cycle. The nested PCR products were then column purified (Qiagen) andcloned into pcDNA3.1 v5/his TOPO vector and sequenced.

One clone, named mkα7-5′N#16, contains the 5′ Met of rhesus monkey alpha7 and is shown below. mkα7-5′N#16: the starting Met is bolded (SEQ IDNO: 16) GAGAGGCGGCTCTGTGGCCACAGGCGCAGGCCCGGGCGACAGCCGATACGTGAGGCGCGCCGGCCCGCGGCAGCTCCGGGACTCAACATGCGCTGCTCGCAGGGAGGCGTCTGGCTGGCTCTGGCCGCGTCGCTCCTGCATGTGTCCCTGCAAGGCGAGTTCCAGAGGAAGCTTTACAAGGAGCTGGTCAAGAACTACAACCCCTTGGAGAGGCCCGTGGCCAATGACTCGCAACCGCTCACCGTCTAC

Full-length Cloning of Rhesus Monkey Nicotinic Alpha-7 Subunit

Two primers, mkα7-5′b and mkα7-3′a, were designed based on the sequencesof mKα7-5′N#16 and mk-3′N#2 (see above) and used to PCR full-lengthrhesus monkey alpha-7 from the same cDNA library. (SEQ ID NO: 17)mkα7-5′b: 5′-CTCAACATGCGCTGCTCGCAGGGAGG (SEQ ID NO: 18) mkα7-3′a:5′-CCAAGCCAGAGGCCTTGCCCATCTGTGAG

The PCR reaction was performed as described above with the followingcycling characteristics: 94° C. for 5′ for 1 cycle; 94° C. for 30seconds, 65° C. for 30 seconds and 68° C. for 2 minutes for 35 cycles;68° C. for 7 minutes for 1 cycle. The resulting PCR fragment (˜1.6 kb)was column purified (Qiagen) and cloned into pcDNA3.1 v5/his TOPO vectorand sequenced.

One clone, named mkalpha7#11, contained the full-length cDNA sequence ofrhesus monkey alpha 7. The cDNA and protein sequences are shown in FIG.1 (SEQ ID NO: 1) and FIG. 2 (SEQ ID NO: 2), respectively. This clone,with the 5′ Met of the full-length rhesus monkey nicotinic alpha-7 isdownstream of the vector CMV promoter, can be used to establish a stablecell line to expressed the recombinant receptor.

Example 2 Generating a Mutant Rhesus Monkey Alpha 7, mkalpha7(L270T)

To mutagenize rhesus monkey alpha 7 at amino acid position 270, theprimers shown below were used to PCR mkalpha7#11, according to theprotocol of Quick-Change kit (Stratagene). After the reaction, the PCRproducts were treated with DpnI at 37° C. for one hour. After treatment,the remaining products were used to transform Top10 competent cells(Invitrogen). Plasmid DNAs from three individual clones were sequencedto detect mutation L270T. The nucleic acid and protein sequences of onemutant clone, named mka7L270T#3, are shown in FIG. 3 (SEQ ID NO: 3) andFIG. 4 (SEQ ID NO: 4), respectively. Primers: (SEQ ID NO: 19) mkα7-14:5′-GGGATAACAGTCACTTCTCTCACTGTCTTC (SEQ ID NO: 20) mkα7-15:5′-GAAGACAGTGAGAGAAGTGACTGTTATCCC

Generating a Double-mutant Rhesus Monkey Alpha 7, mkalpha7(L270T/S193N)

To mutagenize rhesus monkey alpha 7 at amino acid position 193, inaddition to position 270, the following primers (see below) were used toPCR mka7L270T#3, according to the protocol of Quick-Change kit(Stratagene). After the reaction, the PCR products were treated withDpnI at 37° C. for one hour. After treatment, the remaining productswere used to transform Top10 competent cells (Invitrogen). Plasmid DNAsfrom three individual clones were sequenced to detect mutation S193N.The sequences of one double-mutant clone, named mka7L270T/S193N#1, areshown in FIGS. 5 and 6. mkalpha7S193N-5′: (SEQ ID NO: 21)5′-AGTGGCTATATCCCCAACGGAGAATGGGACCTAG 3′ mkalpha7S193N-3′: (SEQ ID NO:22) 5′-CTAGGTCCCATTCTCCGTTGGGGATATAGCCAC 3′

Example 3 Cell Culture and Transfections

QM 7 cells were routinely grown as monolayers in minimum essentialmedium 199 supplemented with 5% FBS, 2% tryptose phosphate broth, 1%DMSO, penicillin (100 IU/ml) and were passaged every 3-4 days.Transfections with either rhesus monkey wild type alpha7 nAchR (mkα7) ormutant alpha7 nAchR (mkα7/L270T) were performed using Lipofectamine PLUSKit (Invitrogen). Twenty-four hours after the transfection, cells weresplit for stable selection with 1 mg/ml G418. Single colonies wereisolated and propagated. The expression of mkα7/L270T was identifiedwith FLIPR for fluorescence changes after addition of agonist. Theexpression of mkα7 was identified by radioligand binding to[³H]-methyllycaconitine (Virginio, C., A., Giacometti, A. et al. (2002).Pharmacological properties of rat alpha7 nicotinic receptors expressedin native and recombinant cell systems, Eur, J. Pharmacol. 445(3),153-161.)

The double-mutant rhesus monkey alpha 7, mkalpha7(L270T/S 193N),containing plasmids were transiently transfected into QM-7 cells withLipofectamine PLUS reagent (Invitrogen), in 175 cm² flasks. Twenty fourhours after the transfection, the cells were collected and re-seededinto a 96-well plate at a density of 40,000 cells/well for functionalmeasurement.

Example 4 Functional Measurement of Mutant (L270T) mkα7/L270T Receptorand Double Mutant mkalpha7(L270T/S193N) Using FLIPR

Cells were seeded into 96 well black wall and clear-bottom plates(Costar) coated with poly-D-lysine at a density of 20,000 cells per welland cultured overnight. On the second day, the cells were incubated with1×HBSS, 20 mM HEPES, 2.5 mM Probenecid (Sigma), 0.1% BSA, pH 7.4 buffer(FLIPR buffer) in the presence of Fluo-3AM (Molecular Probe) at 37° C.for 60 min. After washing three times to remove the Fluo-3AM, FLIPRbuffer was added into the cell plates. The plates were placed onto aFLIPR (Molecular Devices). Compound addition was performed by FLIPRpipetting system. The fluorescence was monitored (λ_(ex)=488 nM,λ_(EM)=540 nM) for three minutes immediately after the compoundaddition. The relative fluorescence unit (RFU) was measured as peakfluorescence intensity minus basal fluorescence intensity. Curve fittingand parameter estimation were carried out using Graph Pad Prism 3.00(GraphPad Software Inc., California, U.S.A.) The responses to nicotineand GTS-21, nicotinic alpha 7 agonists, for mkα7/L270T Receptor weretested and shown in FIG. 7. The responses of double mutant mka (L270T/S193N) to GTS-21, a nicotinic alpha 7 agonist, were tested and shown inFIG. 9.

Example 5 Functional Measurement of mkα7 Using Patch Clamp Method

Recording

Stably transfected QM7 cells grown on cover slips and expressingwild-type rhesus monkey alpha7 receptors were incubated at 37° C. in thepresence of 5% CO₂. Individual cover slips were removed for functionanalysis and placed in a recording buffer consisting of 140 mM NaCl, 5mM KCl, 10 mM HEPES, 2 mM CaCl₂, 1 mM MgCl₂, 100 mM glucose titrated topH 7.3 and perfused at 2-3 ml/min. Borosilicate recording pipettes werefilled with a solution consisting of 140 mM CsCl, 10 mM HEPES, 4 mMNaCl, 4 mM MgCl₂, 2 mM CaCl₂, 5 mM EGTA titrated to pH 7.3. For wholecell recording, pipettes with resistances between 3.0-8.0 MΩ were used.Upon gigaseal formation, holding potential was set to −60 mV and thecell was ruptured by suction. Cell is held at −60 mV at all times exceptwhere noted below.

Drug Application

DAD-VC perfusion system by ALA Scientific Instruments was modified toinclude an internally perfused pipette to rapidly and precisely deliverdrug (based on Kabokov and Papke, 1998). Briefly, drug flows within thepipette and out to a waste receptacle by gravity. By closing the valvethat leads to the waste container, the drug is forced out of the pipetteonto the cell. Prior to drug application, the cell is hyperpolarized to−100 mV for a duration of 500 ms. During the hyperpolarization, drug israpidly dispensed for 1.5 seconds. Recording continues at −100 mV foranother 4 seconds before returning the cell to the holding potential of60 mV. Upon completion of recording, the perfusion system primes thepipette with the next dosage. An intersweep interval equal to 65 secondsis sufficient to completely exchange the contents of the perfusionpipette. The cell is initially exposed to a concentration of 1 mMAcetylcholine (ACh), which maximally activates alpha7 receptors (Papkeand Porter Papke, 2002). Two experimental drug concentrations follow andthe protocol concludes with one final dose of 1 mM ACh.

Data Analysis

Net charge analysis is used to assess the total amount of charge flowingthrough the channel in response to receptor agonism. Net charge analysisis believed to give a better indication of the nicotinic alpha-7receptor than more traditional peak measurements (Papke and PorterPapke, 2002). Each experimental concentration of drug is compared to thecell's initial response to 1 mM ACh. The final Ach response is also usedto account for any receptor desensitization. All experimental values aretherefore normalized to the cell's maximum response to 1 mM ACh. Thesenormalized values are used to generate a dose response curve thatreflects the efficacy of the alpha7 receptor to either a known orexperimental drug (FIG. 8).

The topic headings set forth above are meant as guidance as to wherecertain information can be found in the application. They are notintended to be the only source in the application where information onsuch a topic can be found.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make changes andmodifications of the invention to adapt it to various usage andconditions.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The preceding preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe remainder of the disclosure in any way whatsoever.

The entire disclosure of all applications, patents and publications,cited above and in the figures are hereby incorporated in their entiretyby reference, including U.S. Provisional Application Ser. Nos.60/447,288, filed Feb. 14, 2003, and 60/453,204, filed Mar. 11, 2003

1. An isolated polynucleotide for a monkey alpha-7 nicotinic receptor,or a mutation thereof.
 2. An isolated polynucleotide for an alpha-7nicotinic acetylcholine receptor, comprising: (a) a polynucleotidesequence set forth in FIG. 1 (SEQ ID NO: 1), (b) a polynucleotidesequence coding for a polypeptide having the amino acid sequence fromamino acid 1-502 or 23-502 as set forth in FIG. 2 (SEQ ID NO: 2), (c) apolynucleotide sequence coding for a polypeptide having one or moreamino acid substitutions in the M2 region of the polypeptide sequenceset forth in FIG. 2 (SEQ ID NO: 2) from amino acid 1-502 or 23-502, orcomplements thereto.
 3. An isolated polynucleotide of claim 1, whichcodes without interruption for said alpha-7 nicotinic acetylcholinereceptor.
 4. An isolated polynucleotide of claim 1, comprising apolynucleotide sequence coding for a polypeptide having one or more ofthe following amino acid substitutions: L270T, L270S, L270F, L270V,V274T, T267Q, E260A/V274T, E260A/L270T, E260A/L270S, E260A/L270V,E260A/L270F, E260A/T267Q, E260A/L277T, and/or E260A/L278T.
 5. Anisolated polynucleotide of claim 4, comprising a polynucleotide sequenceset forth in FIG. 3 (SEQ ID NO : 3), or a polynucleotide sequence codingfor a polypeptide comprising an amino acid substitution L270T as setforth in FIG. 4 (SEQ ID NO : 4).
 6. An isolated polynucleotide of claim1 or 2, comprising a polynucleotide sequence coding for a polypeptidecomprising one or more of the following amino acid substitutions: A65V,R156W, S193N, K208R, K208S, M395V, A398V or A398T.
 7. An isolatedpolynucleotide of claim 6, comprising a polynucleotide sequence setforth in FIG. 5 (SEQ ID NO: 5), or a polynucleotide sequence coding fora polypeptide comprising amino acid substitutions L270T and S193N as setforth in FIG. 6 (SEQ ID NO: 6).
 8. An isolated polynucleotide of claim1, wherein said polynucleotide has the complete polynucleotide sequencecoding for monkey alpha-7 nicotinic receptor of the cDNA clone containedin the plasmid deposited with the ATCC as deposit PTA-5004.
 9. Anexpression vector, comprising: a polynucleotide of claim 1 or 2 operablylinked to a promoter sequence.
 10. A transfected host cell, comprising:a polynucleotide of claim 1 or
 2. 11. A transfected host cell,comprising: an expression vector comprising a polynucleotide of claim 1or 2 operably linked to a promoter sequence.
 12. A transfected host cellof claim 9, wherein the alpha-7 nicotinic acetylcholine receptor codedfor by said polynucleotide is expressed on the surface membrane of saidhost cell.
 13. A transfected host cell of claim 8, wherein said cell isa QM7 or QT6 cell.
 14. An isolated polypeptide which is coded for apolynucleotide sequence of claim 1 or
 2. 15. A method for identifying anagent that modulates the expression or activity of an alpha-7 nicotinicacetylcholine receptor in transfected host cells, comprising: contactinga transfected host cell of claim 10 with a test agent under conditionseffective for said test agent to modulate the expression or activity ofsaid alpha-7 nicotinic acetylcholine receptor, wherein said receptor isexpressed on the surface membrane of said cell, and determining whethersaid test agent modulates the expression or activity of said alpha- 7nicotinic acetylcholine receptor.
 16. A method of claim 15, wherein saiddetermining comprises: measuring the total amount of charge flowingacross the membrane of said cell, or measuring the change in acalcium-sensitive dye present in said cell, in response to said agent.17. A method of claim 15, wherein the agent activates the expression oractivity of said receptor.
 18. An antibody which is specific for apolypeptide which is coded for by a polynucleotide of claim 1 or
 2. 19.A non-human transgenic mammal comprising, a polynucleotide of claim 1 or2 coding for a monkey alpha-7 nicotinic receptor.
 20. A mammalian cellwhose genome comprises a functional disruption of the endogenous monkeyalpha-7 nicotinic receptor encoding a polynucleotide of claim 1 or 2.21. A method of selecting a polynucleotide sequence or polypeptidecoding for an alpha-7 nicotinic acetylcholine receptor from a databasecomprising polynucleotide or polypeptide sequences, comprisingdisplaying, in a computer-readable medium, a polynucleotide sequence orpolypeptide sequence of claim 1 or 2, wherein said displayed sequenceshave been retrieved from said database upon selection by a user.